Human Platelets Contain and Release an Active ADAMTS-13-Like Metalloprotease.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3527-3527
Author(s):  
Li Liu ◽  
Huiwan Choi ◽  
Bernardo Aubrey ◽  
Angila Bergeron ◽  
Leticia Nolasco ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Platelet Activation ◽  
Fluid Shear Stress ◽  
Vascular Endothelial Cells ◽  
Severe Infection ◽  
Surface Expression ◽  
Human Platelets ◽  
Platelet Surface ◽  
The Difference ◽  
Adhesion Ligand

Abstract The adhesion ligand von Willebrand factor (VWF) is synthesized and stored in vascular endothelial cells and megakaryocytes/platelets. These cells release VWF in response to similar stimulations. However, a longstanding observation stated that, unlike endothelial cells, platelets do not release the ultra-large form (UL) of VWF upon activation. The lack of ULVWF release may be attributed to the difference in multimerization process in the storage granules or the intrinsic proteolysis of the hyperreactive ULVWF. To examine these possibilities, we analyzed the multimer compositions of VWF stored in and released from platelets. We found that ULVWF was detected by immunobloting of washed platelet lysates in the presence, but not in the absence of 5 mM EDTA. Similarly, when stimulated with 5 μM of thrombin receptor-activating peptide (TRAP), platelets released only VWF that was similar in sizes to plasma VWF, suggesting that platelet ULVWF may have been cleaved before or during its release. In support of this hypothesis, we found that platelets expressed ADAMTS-13-like molecule by western blot of platelet lysates and flow cytometry using two different ADAMTS-13 antibodies. The surface expression of ADAMTS-13-like molecule increased significantly upon platelet activation by TRAP and ADP. Consistent with these observations, the Triton-X-100 lysates of washed platelets and supernatant of washed platelets activated by TRAP contained VWF-cleaving activity, as demonstrated by the cleavage of ULVWF strings formed on histamine-stimulated endothelial cells under fluid shear stress. Our finding is consistent with a previous report, but further showed that this ADAMTS-13-like molecule exists not only in cytoplasma, but also on the surface of platelets. More importantly, the ADAMTS-13-like molecule increased its expression on platelet surface and was released upon platelet activation. The ADAMTS-13-like molecule either in platelets or released upon activation was active in cleaving ULVWF. This intrinsic mechanism for ULVWF proteolysis may be physiologically important. It may prevent the sudden release of hyper-reactive ULVWF from platelets and serve as the second pool of ADAMTS-13 to encounter the increase in ULVWF release from endothelial cells during conditions such as severe infection. The role of the platelet membrane-bound ADAMTS-13 in hemostasis remains to be further determined.

Regulated Shedding of sema4D from the Platelet Surface Produces a Bioactive Second Messenger in Thrombotic Disorders.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1641-1641
Author(s):  
Li Zhu ◽  
Wolfgang Bergmeier ◽  
Jie Wu ◽  
Hong Jiang ◽  
Nana Yeboah ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Platelet Activation ◽  
Transmembrane Domain ◽  
Thrombus Formation ◽  
Active Form ◽  
Extracellular Domain ◽  
Human Platelets ◽  
Biologically Active ◽  
Western Blots ◽  
Platelet Surface

Abstract Proteins that are expressed on the platelet surface can participate in contact-dependent signaling events which modulate thrombus formation or, after being shed from the platelet surface, serve as bioactive messengers that affect the function of nearby cells. Here we show for the first time that platelets express the class IV semaphorin known as sema4D or CD100, and that platelet activation causes the regulated shedding of the sema4D extracellular domain in a biologically-active form. Sema4D is a glycosylated 150 kDa disulfide-linked homodimer that has previously been implicated in interactions between T-cells and B-cells. Platelet activation by collagen, thrombin or PMA causes a transient increase in sema4D surface expression peaking at 15 min, followed by a complete loss of expression over 30–60 minutes. These events are accompanied by the release of the sema4D exodomain as a 130 kDa fragment, leaving a 25–30 kDa transmembrane and cytoplasmic domain fragment that is retained by the platelets. The cleavage event required to produce these fragments is inhibited by metalloprotease inhibitors and abolished in platelets from chimeric mice lacking the metalloprotease known as ADAM17 or TACE (TNF alpha converting enzyme). Incubation of recombinant sema4D with ADAM17 identified a single cleavage site just outside the predicted transmembrane domain. Western blots show that human platelets express ADAM17 in both its immature (zymogen) and mature (active) forms, and indicate that at least some of the ADAM17 is located on the platelet surface. ADAM17-dependent cleavage of sema4D does not require platelet aggregation, but the rate is accelerated when aggregation is allowed to occur and slowed when aggregation is prevented. Under both sets of conditions, cleavage of sema4D occurs to a greater extent and more rapidly than the ADAM17-dependent cleavage of GP Ib alpha, suggesting that there is a hierarchy of proteolytic events when platelets are activated. In terms of biological impact, the shedding of sema4D following platelet activation raises the possibility that the soluble extracellular domain of sema4D serves as a bioactive messenger. Two receptors for sema4D have been identified previously: CD72, which is present on lymphocytes where it regulates the activity of the tyrosine phosphatase, SHP-1, and plexin-B1, which is expressed in endothelial cells. Western blots suggest that both of these receptors are expressed on human platelets and show that SHP-1 is associated with CD72 in resting, but not activated platelets. Taken together, these results demonstrate that sema4D undergoes regulated ADAM17-dependent shedding when platelets are activated, and suggest that this results in the production of a bioactive form of the molecule that can affect responses in nearby platelets, lymphocytes and endothelial cells at sites of thrombosis.

Platelet Activation by Antiphospholipid Antibodies Depends on Epitope Specificity and is Prevented by mTOR Inhibitors

2019 ◽  
Vol 119 (07) ◽  
pp. 1147-1153 ◽  
Author(s):  
Anne Hollerbach ◽  
Nadine Müller-Calleja ◽  
Svenja Ritter ◽  
Friederike Häuser ◽  
Antje Canisius ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Antiphospholipid Antibodies ◽  
Platelet Rich Plasma ◽  
Mtor Inhibitors ◽  
Surface Expression ◽  
Human Platelets ◽  
Antigen Specificity ◽  
Platelet Surface ◽  
Glycoprotein I

AbstractAntiphospholipid antibodies (aPL) have been reported to activate platelets. This is considered to be one of the pathogenic properties of aPL. Even though aPL heterogeneity is quite well established, little is known, if the ability to activate platelets is common to all aPL or depends on antigen specificity. To further study this issue, we analyzed the ability of three human monoclonal aPL with distinctly different antigenic specificities to activate platelets in vitro. The results obtained with human monoclonal aPL were validated with immunoglobulin G (IgG) fractions obtained from patients with antiphospholipid syndrome (APS). A co-factor-independent human monoclonal anticardiolipin aPL had no discernible effect on human platelets. Two monoclonal aPL reactive against β2 glycoprotein I (β2GPI) induced platelet aggregation, integrin αIIbβ3 activation and P-selectin surface expression. These data could be confirmed with patient IgG fractions which could only induce aggregation, if they had anti-β2GPI activity. Anti-β2GPI aPL-induced platelet activation depended on interaction of aPL with the low affinity Fcγ-receptor IIa on the platelet surface. It was completely abolished by pretreatment of platelet-rich plasma with the mechanistic target of rapamycin (mTOR) inhibitors rapamycin or everolimus. This extends previous data showing that mTOR is involved in signaling of anti-β2GPI in monocytes and endothelial cells. In conclusion, anti-β2GPI aPL activate platelets while co-factor-independent anticardiolipin aPL have no effect. mTOR is involved in this signaling process which has implications beyond APS, because so far the role of mTOR signaling in platelets is incompletely explored and requires further study.

HMGB1 binds to activated platelets via the receptor for advanced glycation end products and is present in platelet rich human coronary artery thrombi

2015 ◽  
Vol 114 (11) ◽  
pp. 994-1003 ◽  
Author(s):  
Michael Bode ◽  
David Haenel ◽  
Christoph E. Hagemeyer ◽  
Hannah Seeba ◽  
Daniel Duerschmied ◽  
...  
Keyword(s):  
Coronary Artery ◽  
Platelet Activation ◽  
Knockout Mice ◽  
Surface Expression ◽  
Human Platelets ◽  
Wild Type ◽  
Human Coronary Artery ◽  
Specific Expression ◽  
Platelet Surface ◽  
Activated Platelets

SummaryHigh mobility group box 1 (HMGB1) acts as both a nuclear protein that regulates gene expression, as well as a pro-inflammatory alarmin that is released from necrotic or activated cells. Recently, HMGB1-expression in human atherosclerotic plaques was identified. Therapeutic blockade of HMGB1 reduced the development of diet-induced atherosclerosis in ApoE knockout mice. Thus, we hypothesised an interaction between HMGB1 and activated platelets. Binding of recombinant HMGB1 to platelets was assessed by flow cytometry. HMGB1 bound to thrombin-activated human platelets (MFI 2.49 vs 25.01, p=0.0079). Blood from wild-type, TLR4 and RAGE knockout mice was used to determine potential HMGB1 receptors on platelets. HMGB1 bound to platelets from wild type C57Bl6 (MFI 2.64 vs 20.3, p< 0.05), and TLR4-/- mice (MFI 2.11 vs 25.65, p< 0.05) but failed to show binding to platelets from RAGE-/- mice (p > 0.05). RAGE expression on human platelets was detected by RT-PCR with mRNA extracted from highly purified platelets and confirmed by Western blot and immunofluorescence microscopy. Platelet activation increased RAGE surface expression (MFI 4.85 vs 6.74, p< 0.05). Expression of HMGB1 in human coronary artery thrombi was demonstrated by immunohistochemistry and revealed high expression levels. Platelets bind HMGB1 upon thrombin-induced activation. Platelet specific expression of RAGE could be detected at the mRNA and protein level and is involved in the binding of HMGB1. Furthermore, platelet activation up-regulates platelet surface expression of RAGE. HMGB1 is highly expressed in platelet-rich human coronary artery thrombi pointing towards a central role for HMGB1 in atherothrombosis, thereby suggesting the possibility of platelet targeted anti-inflammatory therapies for atherothrombosis.

Immunological Evidence for Prothrombin in Human Platelets

1986 ◽  
Vol 55 (02) ◽  
pp. 268-270
Author(s):  
R J Alexander
Keyword(s):  
Electrophoretic Mobility ◽  
Platelet Activation ◽  
Double Diffusion ◽  
Human Platelets ◽  
Secreted Proteins ◽  
Platelet Surface ◽  
Activated Platelets ◽  
Similar Protein ◽  
Diffusion Assay ◽  
Supernatant Solution

SummaryAn attempt was made to isolate from plasma the platelet surface substrate for thrombin, glycoprotein V (GPV), because a GPV antigen was reported to be present in plasma (3). Plasma fractionation based on procedures for purification of GPV from platelets revealed a thrombin-sensitive protein with appropriate electrophoretic mobility. The protein was purified; an antiserum against it i) reacted with detergent-solubilized platelet proteins or secreted proteins in a double diffusion assay, ii) adsorbed a protein from the supernatant solution of activated platelets, and iii) inhibited thrombin-induced platelet activation, but the antiserum did not adsorb labeled GPV. The purified protein was immunochemically related to prothrombin rather than to GPV. Other antibodies against prothrombin were also able to adsorb a protein from platelets. It is concluded that 1) plasma does not contain appreciable amounts of GPV, and 2) platelets contain prothrombin or an immunochemically similar protein.

Glycocalyx modulates the motility and proliferative response of vascular endothelium to fluid shear stress

2007 ◽  
Vol 293 (2) ◽  
pp. H1023-H1030 ◽  
Author(s):  
Yu Yao ◽  
Aleksandr Rabodzey ◽  
C. Forbes Dewey
Keyword(s):  
Endothelial Cells ◽  
Vascular Endothelium ◽  
Fluid Shear Stress ◽  
Vascular Endothelial Cells ◽  
Surface Profile ◽  
Cell Junctions ◽  
Vascular Endothelial ◽  
Fluid Shear ◽  
New Perspective ◽  
Cell Cell

Flow-induced mechanotransduction in vascular endothelial cells has been studied over the years with a major focus on putative connections between disturbed flow and atherosclerosis. Recent studies have brought in a new perspective that the glycocalyx, a structure decorating the luminal surface of vascular endothelium, may play an important role in the mechanotransduction. This study reports that modifying the amount of the glycocalyx affects both short-term and long-term shear responses significantly. It is well established that after 24 h of laminar flow, endothelial cells align in the direction of flow and their proliferation is suppressed. We report here that by removing the glycocalyx by using the specific enzyme heparinase III, endothelial cells no longer align under flow after 24 h and they proliferate as if there were no flow present. In addition, confluent endothelial cells respond rapidly to flow by decreasing their migration speed by 40% and increasing the amount of vascular endothelial cadherin in the cell-cell junctions. These responses are not observed in the cells treated with heparinase III. Heparan sulfate proteoglycans (a major component of the glycocalyx) redistribute after 24 h of flow application from a uniform surface profile to a distinct peripheral pattern with most molecules detected above cell-cell junctions. We conclude that the presence of the glycocalyx is necessary for the endothelial cells to respond to fluid shear, and the glycocalyx itself is modulated by the flow. The redistribution of the glycocalyx also appears to serve as a cell-adaptive mechanism by reducing the shear gradients that the cell surface experiences.

scholarly journals von Willebrand factor bound to glycoprotein Ib is cleared from the platelet surface after platelet activation by thrombin

Blood ◽  
1992 ◽  
Vol 79 (8) ◽  
pp. 2011-2021 ◽  
Author(s):  
P Hourdille ◽  
HR Gralnick ◽  
E Heilmann ◽  
A Derlon ◽  
AM Ferrer ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Activation ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Surface Expression ◽  
Von Willebrand Disease ◽  
Platelet Surface ◽  
Immunogold Staining ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract We recently reported that after activation of human platelets by thrombin, glycoprotein (GP) Ib-IX complexes are translocated to the surface-connected canalicular system (SCCS) (Blood 76:1503, 1990). As GPIb is a major receptor for von Willebrand factor (vWF) in platelet adhesion, we have now examined the consequences of thrombin activation on the organization of vWF bound to GPIb on the platelet surface. Studies were performed using monoclonal or polyclonal antibodies in either immunogold staining and electron microscopy (Au-EM) or in flow cytometry. When unstirred platelet-rich plasma was incubated with ristocetin, bound vWF was located by Au-EM as discrete masses regularly distributed over the cell surface. Platelets from a patient with Glanzmann's thrombasthenia, lacking GPIIb-IIIa complexes, gave a similar pattern, confirming that this represented binding to GPIb. That ristocetin was not precipitating vWF before their binding to the platelets was shown by the detection of similar masses on the surface of platelets of a patient with type IIB von Willebrand disease. Experiments were continued using washed normal platelets incubated in Tyrode-EDTA, the purpose of the EDTA being to limit the surface expression of endogenous vWF after platelet stimulation. Under these conditions, platelets were treated with ristocetin for 5 minutes at 37 degrees C in the presence of increasing amounts of purified vWF. This was followed by incubation with thrombin (0.5 U/mL) for periods of up to 10 minutes. Flow cytometry showed a time-dependent loss in the surface expression of vWF bound to GPIb and these changes were confirmed by Au-EM. In particular, immunogold staining performed on ultrathin sections showed that the bulk of the vWF was being cleared to internal membrane systems. Surface clearance of vWF during thrombin- induced platelet activation is a potential mechanism for regulating platelet adhesivity.

scholarly journals Fluid shear stress modulates surface expression of adhesion molecules by endothelial cells

Blood ◽  
1995 ◽  
Vol 85 (7) ◽  
pp. 1696-1703 ◽  
Author(s):  
M Morigi ◽  
C Zoja ◽  
M Figliuzzi ◽  
M Foppolo ◽  
G Micheletti ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Laminar Flow ◽  
Fluid Shear Stress ◽  
Leukocyte Adhesion ◽  
Interleukin 1 ◽  
Static Condition ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Interleukin 1 Beta

We investigated the effect of hemodynamic shear forces on the expression of adhesive molecules, E-selectin, and intercellular adhesion molecule-1 (ICAM-1) on human umbilical vein endothelial cells (HUVEC) exposed to laminar (8 dynes/cm2) or turbulent shear stress (8.6 dynes/cm2 average), or to a static condition. Laminar flow induced a significant time-dependent increase in the surface expression of ICAM-1, as documented by flow cytometry studies. Endothelial cell surface expression of ICAM-1 in supernatants of HUVEC exposed to laminar flow was not modified, excluding the possibility that HUVEC exposed to laminar flow synthetize factors that upregulate ICAM-1. The effect of laminar flow was specific for ICAM-1, while E-selectin expression was not modulated by the flow condition. Turbulent flow did not affect surface expression of either E-selectin or ICAM-1. To evaluate the functional significance of the laminar-flow-induced increase in ICAM-1 expression, we studied the dynamic interaction of total leukocyte suspension with HUVEC exposed to laminar flow (8 dynes/cm2 for 6 hours) in a parallel-plate flow chamber or to static condition. Leukocyte adhesion to HUVEC pre-exposed to flow was significantly enhanced, compared with HUVEC maintained in static condition (233 +/- 67 v 43 +/- 16 leukocytes/mm2, respectively), and comparable with that of interleukin-1 beta treated HUVEC. Mouse monoclonal antibody anti-ICAM-1 completely blocked flow-induced upregulation of leukocyte adhesion. Interleukin-1 beta, which upregulated E-selectin expression, caused leukocyte rolling on HUVEC that was significantly lower on flow- conditioned HUVEC and almost absent on untreated static endothelial cells. Thus, laminar flow directly and selectively upregulates ICAM-1 expression on the surface of endothelial cells and promotes leukocyte adhesion. These data are relevant to the current understanding of basic mechanisms that govern local inflammatory reactions and tissue injury.

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