scholarly journals Thrombin-induced GPIb-IX centralization on the platelet surface requires actin assembly and myosin II activation

Blood ◽  
1996 ◽  
Vol 87 (2) ◽  
pp. 618-629 ◽  
Author(s):  
TJ Kovacsovics ◽  
JH Hartwig
Keyword(s):  
Actin Filaments ◽  
Myosin Ii ◽  
Surface Expression ◽  
Membrane Skeleton ◽  
Cytosolic Calcium ◽  
Human Platelets ◽  
Actin Binding ◽  
Actin Assembly ◽  
Platelet Surface ◽  
Von Willebrand

In resting platelets, the GPIb-IX complex, the receptor for the von Willebrand factor (vWF), is linked to underlying actin filaments by actin-binding protein (ABP-280). Thrombin stimulation of human platelets leads to a decrease in the surface expression of the GPIb-IX complex, which is redistributed from the platelet surface into the open canalicular system (OCS). Because the centralization of GPIb-IX is inhibited by cytochalasin, it is believed to be linked to actin cytoskeletal rearrangements that take place during platelet activation. We have further characterized the mechanism of GPIb-IX centralization in platelets in suspension. Following thrombin stimulation, GPIb-IX shifts from the membrane skeleton of the resting cell to the cytoskeleton of the activated cell in a reaction sensitive to cytochalasin B. The cytoskeletal association of GPIb-IX involves ABP- 280, as it correlates with the incorporation of ABP-280 into the activated cytoskeleton and because no dissociation of the ABP-280/GPIb- IX complexes is detected after thrombin activation. However, the incorporation of GPIb-IX into the cytoskeleton is complete within 1 minute, whereas GPIb-IX centralization requires 5 to 10 minutes for completion. The movement of GPIb-IX to the cytoskeleton of activated platelets is therefore necessary, but not sufficient for GPIb-IX centralization. Blockage of cytosolic calcium increases induced by thrombin by loading with the cell permeant calcium chelator Quin-2 AM inhibited GPIb-IX centralization by 70%, but did not prevent its association with the activated cytoskeleton. Quin-2 loading did, however, decrease the incorporation of myosin II into the activated cytoskeleton. The role of myosin II was further probed using the myosin light chain kinase (MLCK) inhibitor wortmannin. Wortmannin prevents myosin II association to the activated cytoskeleton and inhibits GPIb- IX centralization by 50%, without affecting actin assembly or the association of GPIb-IX to the cytoskeleton. Only micromolar concentrations of wortmannin, high enough to inhibit MLCK, prevent GPIb- IX centralization. These results indicate that thrombin-induced GPIb-IX centralization requires a minimum of two steps, one associating GPIb-IX to the activated cytoskeleton and the second requiring myosin II activation. The involvement of myosin II implies that GPIb-IX/ABP-280 complexes, linked to actin filaments, are pulled into the cell center, and that platelets may exert contractile tension on vWF bound to its receptor.

scholarly journals Effect of aspirin on platelet-von Willebrand factor surface expression on thrombin and ADP-stimulated platelets

Blood ◽  
1989 ◽  
Vol 74 (6) ◽  
pp. 2016-2021 ◽  
Author(s):  
RI Parker ◽  
HR Gralnick
Keyword(s):  
Von Willebrand Factor ◽  
Shape Change ◽  
Adenosine Diphosphate ◽  
Surface Expression ◽  
Platelet Surface ◽  
Granule Secretion ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Platelet Shape ◽  
Stimulation Of

Abstract Platelets contain a pool of endogenous platelet-von Willebrand factor (vWF) that becomes expressed on the platelet surface when platelets are stimulated by a variety of agonists. Maximal platelet-vWF expression occurs in concert with platelet alpha-granule secretion. Aspirin (ASA) is known to impair platelet activation and alpha-granule secretion by irreversible inhibition of platelet cyclo-oxygenase. We studied native and ASA-treated platelets for their ability to mobilize and to express platelet-vWF in response to adenosine diphosphate (ADP) or thrombin. We found that each agonist was effective in promoting increased platelet- vWF surface expression on native and ASA-treated platelets. ASA-treated platelets responded identically to native platelets to low (0.01 U/mL) and high (1.0 U/mL) concentrations of thrombin, while the ADP-induced increase in ASA-treated platelets was only 50% to 60% of that for control platelets. Measurement of secreted platelet-vWF and beta- thromboglobulin indicated that the increase seen with ADP was largely independent of alpha-granule secretion. Using monoclonal antibodies (MoAbs) against the platelet glycoproteins (GP) IIb/IIIa and Ib (MoAbs 10E5 and 6D1, respectively), we demonstrated that the ADP-induced increase in platelet-vWF expression on control platelets primarily involved the binding of secreted platelet-vWF to the platelet GPIIb/IIIa. In contrast, the increase in platelet-vWF that occurred following ADP stimulation of ASA-treated platelets was largely insensitive to GPIIb/IIIa blockade. No effect of GPIb blockade in platelet-vWf expression was noted for either control or ASA-treated platelets. When platelet shape change was prevented by the addition of cytochalasin D, ADP-induced platelet-vWf surface expression on ASA- treated platelets was reduced by more than 80%. Our data indicate that platelets in which the cyclooxygenase pathway is blocked by the action of aspirin can increase surface expression of platelet-vWf as a consequence of platelet shape change. We speculate that this process exposes platelet-vWf bound to GPIIb/IIIa, or possibly GPIb, within the surface connected canalicular system.

scholarly journals The activation-induced decrease in the platelet surface expression of the glycoprotein Ib-IX complex is reversible

Blood ◽  
1994 ◽  
Vol 83 (12) ◽  
pp. 3562-3573 ◽  
Author(s):  
AD Michelson ◽  
SE Benoit ◽  
MH Kroll ◽  
JM Li ◽  
MJ Rohrer ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Surface Expression ◽  
Platelet Surface ◽  
Kinase C ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Thrombin decreases the platelet surface expression of the glycoprotein (GP) Ib-IX complex. To determine whether this effect is reversible, flow cytometric studies were performed with GPIb-IX-specific monoclonal antibodies. In both whole blood and washed platelet systems, incubation of platelets with thrombin or a combination of adenosine diphosphate and epinephrine resulted in a maximal decrease of the platelet surface expression of GPIb-IX within 5 minutes, after which there was a time- dependent return of the platelet surface GPIb-IX complex, which was maximal by 60 minutes. Exposure of the same platelets to additional exogenous thrombin resulted in a second decrease in platelet surface GPIb-IX, followed by a second reconstitution of platelet surface GPIb- IX. Throughout these experiments there was no measurable release from the platelets of glycocalicin (a proteolytic fragment of GPIb). Experiments in which platelets were preincubated with a biotinylated GPIb-specific MoAb showed that the GPIb molecules that returned to the platelet surface were the same molecules that had been translocated to the intraplatelet pool. The GPIb molecules that returned to the platelet surface were functionally competent to bind von Willebrand factor, as determined by ristocetin-induced platelet agglutination and ristocetin-induced binding of exogenous von Willebrand factor. Inhibitors of protein kinase C and myosin light-chain kinase enhanced the reexpression of platelet surface GPIb. In summary, the activation- induced decrease in the platelet surface expression of the GPIb-IX complex is reversible. Inactivation of protein kinase C and myosin light-chain kinase are important mechanisms in the reexpression of the platelet surface GPIb-IX complex.

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 Neutrophil cathepsin G modulates the platelet surface expression of the glycoprotein (GP) Ib-IX complex by proteolysis of the von Willebrand factor binding site on GPIb alpha and by a cytoskeletal-mediated redistribution of the remainder of the complex

Blood ◽  
1994 ◽  
Vol 84 (1) ◽  
pp. 158-168 ◽  
Author(s):  
CA LaRosa ◽  
MJ Rohrer ◽  
SE Benoit ◽  
MR Barnard ◽  
AD Michelson
Keyword(s):  
Binding Site ◽  
Von Willebrand Factor ◽  
Cytochalasin B ◽  
Surface Expression ◽  
Cathepsin G ◽  
Prostaglandin I2 ◽  
Platelet Surface ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Platelet Content

Abstract The effects of neutrophil cathepsin G on the glycoprotein (GP) Ib-IX complex of washed platelets were examined. Cathepsin G resulted in a concentration- and time-dependent decrease in the platelet surface GPIb- IX complex, as determined by flow cytometry, binding of exogenous von Willebrand factor (vWF) in the presence of ristocetin, and ristocetin- induced platelet agglutination. Cathepsin G resulted in proteolysis of the vWF binding site on GPIb alpha (defined by monoclonal antibody [MoAb] 6D1), as determined by increased supernatant glycocalicin fragment (a proteolytic product of GPIb alpha); decreased total platelet content of GPIb; and lack of effect of either cytochalasin B (an inhibitor of actin polymerization), prostaglandin I2 (an inhibitor of platelet activation), or prior fixation of the platelets. However, cathepsin G resulted in minimal decreases in the binding to fixed platelets of MoAbs TM60 (directed against the thrombin binding site on GPIb alpha) and WM23 (directed against the macroglycopeptide portion of GPIb alpha). In contrast to its proteolytic effect on GPIb alpha, the cathepsin G-induced decrease in platelet surface GPIX and the remnant of the GPIb-IX complex (defined by MoAbs FMC25 and AK1) was via a cytoskeletal-mediated redistribution, as determined by lack of change in the total platelet content of GPIX and the GPIb-IX complex; complete inhibition by cytochalasin B, prostaglandin I2, and prior fixation of platelets. Experiments with Serratia protease-treated and Bernard- Soulier platelets showed that neither platelet surface GPIb nor cathepsin G-induced proteolysis of GPIb were required for the cathepsin G-induced redistribution of the remnant of the GPIb-IX complex or the cathepsin G-induced increase in platelet surface P-selectin. In summary, neutrophil cathepsin G modulates the platelet surface expression of the GPIb-IX complex both by proteolysis of the vWF binding site on GPIb alpha and by a cytoskeletal-mediated redistribution of the remainder of the complex. Prior studies show that, although thrombospondin 1, antiserine proteases, and plasma are all inhibitors of cathepsin G, the effects of cathepsin G on platelets, including an increase in surface GPIIb-IIIa, occur during close contact between neutrophils and platelets in a protective microenvironment (eg, thrombosis and local inflammation).(ABSTRACT TRUNCATED AT 400 WORDS).

Platelet Adhesion Plaques: Anchors for Contraction of the Hemostatic Plug.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 5128-5128
Author(s):  
James G. White ◽  
Steven M. Burris ◽  
Gines Escolar
Keyword(s):  
Actin Filaments ◽  
Conflicts Of Interest ◽  
Membrane Surface ◽  
Protein A ◽  
Human Platelets ◽  
Contractile Force ◽  
Actin Binding ◽  
Clot Retraction ◽  
Rhodamine Phalloidin ◽  
Adhesion Plaques

Abstract Abstract 5128 Exposure of GPIIb-IIIa and other receptors on the surface of activated platelets, binding of fibrinogen, molding of shape-changed cells into tight aggregates, internal assembly of actin molecules into filaments and movement of talin, an actin-binding protein, to the inner membrane surface provides the framework for clot retraction. However, the direction of contractile force towards the center of large aggregates or clots would lift the hemostatic plug away from the edges of vascular injury. Another mechanism must be present to facilitate the direction of contractile force toward the damaged vessel wall. This may be accomplished by development of adhesion plaques as platelets spread out on the injured vessel. The present study has used scanning (SEM) and transmission electron microscopy (TEM), confocal and immunofluorescence microscopy to detect adhesion plaques developing at sites of contact as platelets spread on surfaces. Rhodamine-phalloidin was used to detect actin filaments, and an anti-talin antibody identified by protein-A gold or Alexa Fluor 488 labeled rabbit anti mouse IgG to demonstrate talin. Normal human platelets were spread on clean glass slides or plastic chambers for intervals of up to 90 min, extracted with Triton X100 or fixed intact then labeled for talin and actin, and prepared for study by the several microscopic techniques. Triton-extracted spread platelets revealed attachment plaques well stained for talin and actin when examined by SEM or TEM. Inmunofluorescence studies of spread platelets stained with rhodamine-phalloidin and antibodies also revealed co-participation of actin filaments and talin in formation of the adhesion plaques. The association of actin and talin remained intact at all intervals for up to 90 min. Clearly adhesion plaques serve as the anchors for contraction and sealing of hemostatic plug to damaged vascular surfaces. Disclosures No relevant conflicts of interest to declare.

Analysis of Platelet Receptor Expression in ITP,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3289-3289
Author(s):  
Jianlin Qiao ◽  
Huy Tran ◽  
Fi-Tjen Mu ◽  
Robert K Andrews ◽  
Elizabeth E Gardiner
Keyword(s):  
Platelet Count ◽  
Conflicts Of Interest ◽  
Surface Expression ◽  
Experimental Models ◽  
Receptor Expression ◽  
Response To Treatment ◽  
Platelet Surface ◽  
Healthy Donors ◽  
Platelet Receptor ◽  
Von Willebrand

Abstract Abstract 3289 In this study we assess platelet receptor expression and shedding in patients with immune thrombocytopenia (ITP) before and during treatment. The aim is to evaluate the potential value of quantitative measurement of platelet receptors for diagnosis and/or monitoring treatment in thrombocytopenia due to immune or other causes. The platelet-specific collagen receptor, glycoprotein (GP)VI, is associated with the Fc receptor γ-chain (FcRγ). GPVI/FcRγ is coassociated on platelet surface with the GPIb-IX-V complex; GPIbα of GPIb-IX-V binds von Willebrand factor and other ligands. Our previous studies showed engagement of platelet FcγRIIa by antiplatelet antibodies induced ectodomain shedding of GPVI, generating soluble ectodomain (sGPVI) in plasma. However, apart from one individual with an anti-GPVI antibody, whether anti-platelet antibodies associated with ITP affect GPVI/GPIb expression/shedding has not been addressed. In this study we used flow cytometry and a sGPVI ELISA to assess 1) whether patients with ITP had dysregulated expression/shedding of GPVI or GPIbα, and 2) whether platelet receptor expression changes prior to recovery of platelet count in individuals undergoing treatment for ITP. In 9 ITP patients (mean age=48.6, range 29–79; 6 female) with platelet count 61±9 × 109/L (range, 33–122 × 109/L), GPVI surface expression (GeoMean±SE, 137±17) was lower than healthy controls (274±26; n=17; platelet count 247±13), and sGPVI in patient plasma was significantly higher (39±4 ng/mL) compared to 17 healthy donors (19±3 ng/mL) (P=0.0006). In longitudinal samples analysed at weekly intervals during 2-month treatment with steroids, decreased GPVI surface expression and increased sGPVI in plasma remained essentially unchanged as the platelet count normalized, consistent with persistent anti-platelet antibody. However, while levels of intact platelet GPIbα were significantly reduced in ITP compared to healthy donors (P=0.0053), they approached healthy levels within 1 week of treatment, preceding improvement in platelet count or other measures. GPIbα expression/cleavage has been previously implicated in platelet clearance in experimental models, and our analysis suggests the proteolytic status of human GPIbα may be a novel early marker for evaluating response to treatment in ITP. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The integral membrane protein, ponticulin, acts as a monomer in nucleating actin assembly.

1993 ◽  
Vol 120 (4) ◽  
pp. 909-922 ◽  
Author(s):  
C P Chia ◽  
A Shariff ◽  
S A Savage ◽  
E J Luna
Keyword(s):  
Membrane Protein ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Lipid Vesicles ◽  
Integral Membrane Protein ◽  
Actin Binding ◽  
Actin Assembly ◽  
Nucleation Activity

Ponticulin, an F-actin binding transmembrane glycoprotein in Dictyostelium plasma membranes, was isolated by detergent extraction from cytoskeletons and purified to homogeneity. Ponticulin is an abundant membrane protein, averaging approximately 10(6) copies/cell, with an estimated surface density of approximately 300 per microns2. Ponticulin solubilized in octylglucoside exhibited hydrodynamic properties consistent with a ponticulin monomer in a spherical or slightly ellipsoidal detergent micelle with a total molecular mass of 56 +/- 6 kD. Purified ponticulin nucleated actin polymerization when reconstituted into Dictyostelium lipid vesicles, but not when a number of commercially available lipids and lipid mixtures were substituted for the endogenous lipid. The specific activity was consistent with that expected for a protein comprising 0.7 +/- 0.4%, by mass, of the plasma membrane protein. Ponticulin in octylglucoside micelles bound F-actin but did not nucleate actin assembly. Thus, ponticulin-mediated nucleation activity was sensitive to the lipid environment, a result frequently observed with transmembrane proteins. At most concentrations of Dictyostelium lipid, nucleation activity increased linearly with increasing amounts of ponticulin, suggesting that the nucleating species is a ponticulin monomer. Consistent with previous observations of lateral interactions between actin filaments and Dictyostelium plasma membranes, both ends of ponticulin-nucleated actin filaments appeared to be free for monomer assembly and disassembly. Our results indicate that ponticulin is a major membrane protein in Dictyostelium and that, in the proper lipid matrix, it is sufficient for lateral nucleation of actin assembly. To date, ponticulin is the only integral membrane protein known to directly nucleate actin polymerization.

scholarly journals von Willebrand factor competes with fibrin for occupancy of GPIIb:IIIa on thrombin-stimulated platelets

Blood ◽  
1990 ◽  
Vol 75 (4) ◽  
pp. 889-894 ◽  
Author(s):  
RR Hantgan ◽  
WL Nichols ◽  
ZM Ruggeri
Keyword(s):  
Von Willebrand Factor ◽  
Three Dimensional ◽  
Fibrin Clot ◽  
Human Platelets ◽  
Terminal Region ◽  
Platelet Surface ◽  
Fibrin Network ◽  
Dependent Inhibition ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract We have investigated two major questions related to the molecular basis of interactions between the three-dimensional fibrin network and thrombin-stimulated human platelets. First, what are the roles played by glycoproteins (GP) Ib and IIb:IIIa in linking the fibrin clot tightly to the platelet surface? Second, does von Willebrand factor (vWF) modulate the extent of platelet-fibrin interactions? Quantitative fluorescence microscopy (microfluorimetry) has been used to determine the quantity of fluorescein-labeled fibrin bound to the surface of thrombin-stimulated, gel-filtered platelets (the supernatants of which contained small quantities of vWF) in the presence/absence of receptor- specific and vWF-specific monoclonal antibodies (MoAbs), as well as exogenous vWF. A MoAb specific for the GPIIb:IIIa complex exhibited a concentration-dependent inhibition of fibrin binding, whereas a MoAb specific for GPIb was ineffective in this regard. Similarly, a MoAb that recognizes the N-terminal region of vWF involved in GPIb binding did not influence fibrin binding. In contrast, a MoAb that binds to a C- terminal region of vWF involved in GPIIb:IIIa recognition caused a specific, concentration-dependent increase in the quantity of platelet- bound fibrin. We also found that exogenous vWF caused a concentration- dependent decrease in fibrin binding. These results support the hypothesis that vWF and fibrin, both of which are multimeric adhesive ligands, compete for occupancy of the GPIIb:IIIa complex on thrombin- stimulated platelets.

scholarly journals Interactions of tensin with actin and identification of its three distinct actin-binding domains.

1994 ◽  
Vol 125 (5) ◽  
pp. 1067-1075 ◽  
Author(s):  
S H Lo ◽  
P A Janmey ◽  
J H Hartwig ◽  
L B Chen
Keyword(s):  
Amino Acids ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Sh2 Domain ◽  
Actin Binding ◽  
Molar Ratio ◽  
Actin Assembly ◽  
Binding Domains ◽  
Focal Contacts ◽  
End Capping

Tensin, a 200-kD phosphoprotein of focal contacts, contains sequence homologies to Src (SH2 domain), and several actin-binding proteins. These features suggest that tensin may link the cell membrane to the cytoskeleton and respond directly to tyrosine kinase signalling pathways. Here we identify three distinct actin-binding domains within tensin. Recombinant tensin purified after overexpression by a baculovirus system binds to actin filaments with Kd = 0.1 microM, cross-links actin filaments at a molar ratio of 1:10 (tensin/actin), and retards actin assembly by barbed end capping with Kd = 20 nM. Tensin fragments were constructed and expressed as fusion proteins to map domains having these activities. Three regions from tensin interact with actin: two regions composed of amino acids 1 to 263 and 263 to 463, cosediment with F-actin but do not alter the kinetics of actin assembly; a region composed of amino acids 888-989, with sequence homology to insertin, retards actin polymerization. A claw-shaped tensin dimer would have six potential actin-binding sites and could embrace the ends of two actin filaments at focal contacts.

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.

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