GPIbα Activation Triggers Platelet Tissue Factor/FVIIa-Dependent Procoagulant Activity, Which Is Dampened By Platelet Secreted TFPI and Protein S. Novel Platelet-Based Model of Hemostasis

Abstract Human platelets contain Tissue Factor (TF) demonstrated by western blotting (WB), IP, confocal IF microscopy and flow cytometry (FC) using an array of different α-TF antibodies. Moreover, TF is synthesized by platelets and has procoagulant activity (PCA) (Panes et al. Blood 2007). Platelets also contain the full-length α-isoform of TFPI that is exposed on the plasma membrane of activated, coated-platelets. A new role of Protein S (PS) is to function as TFPI co-factor for TF/FVIIa/FXa inactivation. In fact, TFPI function, enhanced by protein S (PS), dampens thrombin generation on the platelet surface (Wood, JP et al, 2014). In endothelial cells TFPI appears associated with the cell surface through glycosylphosphatidyl-inositol-mediated anchorage, suggesting some type of association with cholesterol-rich domains in cell membranes (lipid rafts, LR). Platelets also contain PS, but its functional association with platelet TFPI to inhibit platelet TF-dependent PCA remains still unknown. Aim: to disclose the physiologic role of TFPI and PS on the TF-dependent PCA of human platelets. Results: Stimulation of isolated, washed platelets with VWF-Ristocetin (VWF-R) resulted in 10-fold increase of TF-dependent FXa generation within 2-5min, compared with non-stimulated (N-S) platelets: [median 17(10-37) to 182(43-847) nM FXa/2x107 platelets, n=198]. VWF-R induced anionic phospholipid exposure, but no α-δ-granules release in washed platelets. FX activation could be triggered without external FVII, although this was required to sustain the reaction. FVII/FVIIa was demonstrated in washed platelets by WB, confocal IF microscopy and FC, and its membrane expression augmented after platelet activation. FXa induced by VWF-R was abolished by pre-incubation with TFPI or with several polyclonal or mAbs against to each TF, FVIIa or GPIbα. In contrast, TRAP stimulation (n=84) induced little or no FXa generation [Median 32(11-219) nM FXa/2x107platelets], but FX activation was increased by 54% (range 13-109%, p=0.0039) in platelets pre-incubated with α-TFPI. This increase was more pronounced with pre-incubation with α-PS (90%, range 31-227%, p<0.004). Combination of α-TFPI and α-PS did not enhance further FX activation. The releasate fraction of TRAP-stimulated platelets inhibited the FXa produced by VWF-R-stimulated platelets, supporting that TRAP-induced secretion of TFPI and PS explained this effect. Membrane exposure of platelet TFPI measured by FC was decreased after TRAP stimulation, a paradox explained by the high content of TFPI and PS in microparticles (MP) contained in the TRAP releasates. In contrast, the smaller number of MP released by VWF-R activation contained neither TFPI nor PS (WB assay). IP assays in platelet membrane fractions showed co-precipitation of TF, FVII/FVIIa and GPIbα. All these proteins co-precipitated also with TFPI in lipid raft fractions. Importantly, TFPI was notably augmented in LR fractions after TRAP stimulation, as compared with VWF-R stimulation. Moreover, TRAP stimulation resulted in co-precipitation of TFPI and PS in cytosolic, membrane and released platelet fractions. Conclusions: 1. TF-dependent PCA of washed human platelets is specifically and rapidly induced by GPIbα activation and it´s not accompanied with α-δ-granules release, including TFPI and PS. 2. Platelets contain enough membrane FVII to trigger the FX activation. 3. TRAP induces granule release, including TFPI and PS, which block TF-dependent PCA. 3. This TFPI is predominantly localized in LR fractions and co-precipitates with PS. 4. Secreted protein S likely localizes TFPI to the platelet membrane rich in anionic-rich phospholipids. 5. These results suggest that TF is translocated to LR for its inactivation. 6.These findings lead us to propose a novel model in which clotting is triggered by platelet TF during the first stage of platelet adhesion through GPIbα-VWF interaction; and TF/FVIIa/FXa would be dampened by the secreted TFPI and PS during subsequent platelet activation. Human platelets TF, FVII and the anionic phospholipid surface become central players to assemble and localize the whole clotting process into and around the platelet plug for both hemostasis and atherothrombosis; and platelet TFPI and PS would modulate its growth inactivating TF/FVIIa/FXa on the platelet surface. Disclosures No relevant conflicts of interest to declare.

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Platelet Tissue Factor: Fast and Specific Clotting Activation Pathway Mediated by VWF-GPIba Interaction and Platelet Membrane FVII. Human Platelets Contain All the Components for Assembling the Prothrombinase Complex and Their Procoagulant Function Is Independent of Aggregation/Secretion and GPVI Function.

Blood ◽

10.1182/blood.v114.22.3000.3000 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3000-3000

Author(s):

Olga Panes ◽

Valeria Matus ◽

Claudia G. Sáaez ◽

Jaime Pereira ◽

Diego Mezzano

Keyword(s):

Flow Cytometry ◽

Tissue Factor ◽

Platelet Activation ◽

Conflicts Of Interest ◽

Human Platelets ◽

Platelet Membrane ◽

Prothrombinase Complex ◽

Serotonin Secretion ◽

Trap Activation

Abstract Abstract 3000 Poster Board II-977 Human platelets synthesize and store functionally silent tissue factor (TF) which expresses procoagulant activity (PCA) after platelet activation. Fast activation of TF was elicited by VWF-Ristocetin (VWF-R) through GPIbαa activation and Src-Lyn transduction pathway (Blood, Nov 2008; 112:113). Given that GPVI, along with GPIb and TF have been found in “lipid rafts”, and the activated form of GPVI signals through Fyn, another member of the Src family, we tested if GPVI was involved in TF-initiated PCA. We also studied the time-course and pathway specificity of TF activation and the role of platelet FVII in PCA. Weak TF immunofluorescence and co-localization with GPIba were observed in non stimulated washed platelets. A mild increase of TF fluorescence was detected 2 min after TRAP activation, which augmented when the stimulus was VWF-R. Furthermore, striking enhancement of TF fluorescence occurred 2 min after depositing platelets over a VWF-coated surface, but not over fibrinogen or albumin. Platelets adherent to VWF matrix showed GPIb clustering and loss of co-localization with TF. Externalization of TF was confirmed by immunoprecipitation (Ip) of biotinylated membranes before and after platelet activation. Concomitantly, TF-dependent FXa generation increased 5-10-fold shortly after VWF stimulus. Washed platelets stimulated with VWF-R agglutinated normally when stirred in an aggregometer, and the fraction of platelets exposing anionic phospholipids (annexin V binding) was similar to parallel samples stimulated with TRAP. However, VWF-R induced null 14C-serotonin secretion and P-selectin exposure (flow cytometry) in washed platelets. In contrast, TRAP, collagen, ADP and convulxin induced full platelet aggregation, 14C-serotonin and P-selectin secretion at 2-5 min, but with no increase in FXa generation. Platelet PCA was inhibited by antibodies against TF, GPIba, FVIIa, as well as by SU6656 and PP2 (Src pathway inhibitors), but not by Gö6850 (a PKC inhibitor) or exogenous TFPI. p85, a subunit of PI-3K constitutively associated with GPIb complex, becomes strongly associated with TF after stimulation with VWF-R, though only weakly after TRAP activation, confirming the coordinate activation of GPIb and TF. FVII and FX were revealed in platelet membrane fractions by immunoblotting and both co-precipitate with TF in non-stimulated platelets. Two min after activation with VWF-R striking co-precipitations of TF with FVII and FX light chains were evidenced, denoting activation of platelet FVII and FX. When exogenous FX was added to the assay, the amount of FXa generated after 1 and 2 min stimulation was similar whether or not exogenous FVIIa was added. Platelets from four non-related patients with bleeding related to hereditary defect of GPVI had null aggregation and secretion with convulxin and collagen, less than 7% labeling of GPVI by flow cytometry and an immunoreactive membrane GPVI of Mr≈40kDa (native GPVI Mr=62kDa). All of them had normal agglutination with VWF-R and normal FXa generation. In summary, GPIb activation by VWF constitutes a unique and fast inducer of platelet TF-dependent PCA. This process requires anionic phospholipid exposure, but is independent of platelet GPIIb/IIIa and GPVI function. Platelet FVII can initiate FXa generation without need of plasma FVII. The associations of platelet FVII and FX with TF on membrane fractions, together with the large amount of FV in platelets, indicate that human platelets provide not just TF and a PCA phospholipid platform, but also all the components of the prothrombinase complex to trigger the clotting process. Taken together, our results underline the central role of platelets in the whole hemostatic process, unifying primary and secondary hemostasis and circumscribing thrombin generation and fibrin deposition where platelet plug is being formed. Platelet PCA should become a pharmacological target for preventing or managing bleeding and thrombotic disorders. Disclosures: No relevant conflicts of interest to declare.

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Platelet membrane topography: colocalization of thrombospondin and fibrinogen with the glycoprotein IIb-IIIa complex

Blood ◽

10.1182/blood.v66.4.926.926 ◽

1985 ◽

Vol 66 (4) ◽

pp. 926-934 ◽

Cited By ~ 32

Author(s):

AS Asch ◽

LL Leung ◽

MJ Polley ◽

RL Nachman

Keyword(s):

Thin Section ◽

Platelet Activation ◽

Immunoelectron Microscopy ◽

Human Platelets ◽

The Body ◽

Platelet Membrane ◽

Macromolecular Complex ◽

Platelet Surface ◽

Membrane Topography

Abstract The distribution of platelet thrombospondin (TSP), fibrinogen, and glycoproteins IIb-IIIa (GPIIb-IIIa) and GPIb were studied in resting and activated human platelets using frozen thin-section immunoelectron microscopy. In resting platelets, TSP and fibrinogen were found within alpha granules and not on the platelet surface. In unstimulated platelets, GPIIb-IIIa and GPIb were distributed diffusely over the platelet membrane as well as within the body of the platelets. Upon thrombin or A23187 stimulation, TSP, fibrinogen, and GPIIb-IIIa colocalized on the platelet membrane and the canalicular system as well as on pseudopodia and between adherent platelets. GPIb distribution was unchanged by platelet activation. The findings support the hypothesis that a macromolecular complex of TSP-fibrinogen and GPIIb-IIIa forms on the activated platelet membrane.

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Platelet membrane topography: colocalization of thrombospondin and fibrinogen with the glycoprotein IIb-IIIa complex

Blood ◽

10.1182/blood.v66.4.926.bloodjournal664926 ◽

1985 ◽

Vol 66 (4) ◽

pp. 926-934 ◽

Cited By ~ 1

Author(s):

AS Asch ◽

LL Leung ◽

MJ Polley ◽

RL Nachman

Keyword(s):

Thin Section ◽

Platelet Activation ◽

Immunoelectron Microscopy ◽

Human Platelets ◽

The Body ◽

Platelet Membrane ◽

Macromolecular Complex ◽

Platelet Surface ◽

Membrane Topography

The distribution of platelet thrombospondin (TSP), fibrinogen, and glycoproteins IIb-IIIa (GPIIb-IIIa) and GPIb were studied in resting and activated human platelets using frozen thin-section immunoelectron microscopy. In resting platelets, TSP and fibrinogen were found within alpha granules and not on the platelet surface. In unstimulated platelets, GPIIb-IIIa and GPIb were distributed diffusely over the platelet membrane as well as within the body of the platelets. Upon thrombin or A23187 stimulation, TSP, fibrinogen, and GPIIb-IIIa colocalized on the platelet membrane and the canalicular system as well as on pseudopodia and between adherent platelets. GPIb distribution was unchanged by platelet activation. The findings support the hypothesis that a macromolecular complex of TSP-fibrinogen and GPIIb-IIIa forms on the activated platelet membrane.

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Platelet Tissue Factor: Expression of Pro-Coagulant Activity depends on Gpibα Activation and Signaling through Lyn-Mediated Phosphorylation. A Platelet-Based Model of Hemostasis

Blood ◽

10.1182/blood.v112.11.113.113 ◽

2008 ◽

Vol 112 (11) ◽

pp. 113-113

Author(s):

Olga Panes ◽

Paula Ibarra ◽

Valeria Matus ◽

Claudia G. Sáez ◽

Jaime Pereira ◽

...

Keyword(s):

Platelet Aggregation ◽

Tissue Factor ◽

Platelet Activation ◽

Human Platelets ◽

Serine Phosphorylation ◽

Ionophore A23187 ◽

Clotting Time ◽

Sh Groups ◽

Platelet Membranes

Abstract Human platelets synthesize and store functionally silent tissue factor (TF) which has procoagulant activity (PCA) after platelet activation. We now explored the location of inactive and active forms of TF and mechanisms of activation. We reported that resting, non-permeabilized human platelets express scant surface TF, which was strikingly enhanced and co-localized with GPIbα after stimulation (Blood2007;109:5242). The externalization of TF was confirmed by immunoprecipitation (Ip) from biotinylated membranes before and after platelet activation. Moreover, TF and GPIbα co-precipitated in Ip assays and both glycoproteins were prominently found in lipid rafts (Lr) domains. TF-dependent PCA, assessed by FXa generation, was negligible or absent in resting, leukocyte-free platelet preparations. Interestingly, FVII was found in platelet membranes (western blot) and no exogenous FVIIa was needed to trigger TF-dependent FXa generation from washed platelets. Platelet responses to activation (TF-dependent PCA, platelet aggregation and secretion) depended on the agonist used. With 1IU/mL VWF + 1.2 mg/mL Ristocetin (Ris) PCA increased 5 to 10-fold 2 min after stimulation and platelets formed large aggregates with null 14C-serotonin secretion. In contrast, both platelet aggregation and secretion were normal 2 min after activation with 1IU thrombin, 10μM TRAP or 2μg/mL collagen; and PCA induced by thrombin was only ≈1/2 of that elicited by VWF+Ris, whereas 2 min after TRAP or collagen stimulation no PCA induction was detected. Removal of membrane cholesterol (by methyl-β-cyclo-dextrin) or disruption of Lr (with filipin III) abolishes the VWF-Ris-induced PCA. Lr’s from resting platelets contain TF of Mr ≈60kDa. Five min after activation an increase in Lr’s TF was observed, but its Mr depended on the agonist: species of ≈47kDa and ≈60kDa were found after VWF-Ris and TRAP activation, respectively. Given that GPIbα signals through Lyn, member of the Src family, inhibition of signaling with PP2 resulted in 80% fall of VWF-Ris-induced PCA. Ip assays revealed that Lyn co-precipitated with both GPIbα and TF in VWF-Ris activated, but not resting platelets. The phosphokinase activity of Lyn on TF was tested. A polyclonal antibody raised against the phosphorylated (Ser253/Ser258) cytoplasmic domain of TF (Dr. W. Ruf, La Jolla) recognized membrane TF only in activated, not in resting platelets. These findings indicate that VWF-induced activation of GPIbα, subsequent signaling with Lyn-induced serine phosphorylation, along with a change of TF to a ≈47kDa species, triggers human platelets PCA. The previously described role of phospho-disulphide isomerases (PDI) in TF activation through SH groups oxidation was also explored. TF and PDI co-precipitated in resting and activated platelet membranes and antagonizing PDI with bacitracin inhibited TF-dependent PCA. After platelet activation and labeling with MPB, a sulfhydryl-specific probe, TF protein (with PCA) was detected on the plasma membrane, denoting presence of reduced thiols. Furthermore, platelet incubation with phenylarsine oxide, a blocking reagent of vicinal SH groups, or HgCl2, a potent oxidant of thiol groups, had no effect on platelet PCA. Thus, it seems unlikely that TF activation depends on SH oxidation. Finally, we found that platelet TF was sufficient to speed up the clotting of plasma. In fact, clotting time of PRP (2 × 108 platelets mL−1) incubated with ionophore A23187 (2 min, 37°C) and then re-calcified, was 59 ± 6 sec, whereas clotting time in re-calcified PRP without previous activation was 137 ± 19 sec (n=19, p<0.0001). Taken together, our results highlight the crucial role of platelets, not only in assembling clotting complexes and reactions on their surface, but also providing enough TF to trigger the whole process. This novel, comprehensive understanding of hemostasis, (“platelet-based hemostasis model”), unifies primary and secondary hemostasis around the platelets, which would be able to synchronize and modulate the times of both processes ensuring a confined thrombin generation and adequate deposit of fibrin when and where it is needed. It also emphasizes the self-sufficiency of intravascular components to carry out both normal hemostasis and thrombus formation. In this context, platelet PCA may become a central pharmacological target for preventing or managing bleeding and thrombotic disorders.

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Immunological Evidence for Prothrombin in Human Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661534 ◽

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.

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Abstract 11947: Unraveling the Mechanism for the Stimulatory Role of Platelet GPIb-IX-V in Thrombin-Induced Platelet Activation

Circulation ◽

10.1161/circ.130.suppl_2.11947 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Brian Estevez ◽

Michael K Delaney ◽

Aleksandra Stojanovic-Terpo ◽

Xiaoping Du

Keyword(s):

Platelet Activation ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Human Platelets ◽

Platelet Glycoprotein ◽

Inhibitory Peptide ◽

Protease Activated Receptors ◽

Signaling Mechanism ◽

Ovary Cells

Numerous reports indicate that the platelet glycoprotein (GP) Ib-IX complex (GPIb-IX) binds directly to the potent platelet agonist thrombin and is important for promoting thrombin-induced platelet activation. However, how GPIb-IX contributes to thrombin-induced platelet activation is unclear. It has been suggested that thrombin binding to GPIb facilitates the cleavage, and thus activation, of the protease-activated receptors (PAR). Our data indicate that GPIb-IX promotes thrombin signaling through a GPIb-IX signaling mechanism. Pretreatment of human platelets with MPalphaC, an inhibitory peptide based on a critical 14-3-3 signaling protein binding site on the cytoplasmic domain of the GPIb alpha chain, inhibited thrombin-induced platelet activation. MPalphaC-treatment inhibited thrombin-induced activation of Rac1 and LIMK1, both of which are known to play essential roles in GPIb signaling. To more specifically determine the role of GPIb-IX, we reconstituted GPIb-IX-facilitated thrombin signaling in Chinese Hamster Ovary cells expressing PAR1. Thrombin induced signaling was significantly enhanced by GPIb-expression, and deletion of the cytoplasmic 14-3-3-binding domain of GPIb alpha abolished the stimulatory effect of GPIb on thrombin signaling. Furthermore, the role of GPIb-IX in promoting thrombin signaling requires Rac1, and GPIb-IX-dependent Rac1 activation and LIMK phosphorylation are abolished in delta 605 cells expressing a 14-3-3-binding defective mutant GPIb alpha. Taken together, these data suggest that the stimulatory role of GPIb in thrombin signaling requires a C-terminal 14-3-3-binding region which mediates activation of a Rac1/LIMK1 pathway that promotes thrombin signaling leading to platelet activation.

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Collagen Receptor Signaling in Platelets and Megakaryocytes

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615855 ◽

1999 ◽

Vol 82 (08) ◽

pp. 365-376 ◽

Cited By ~ 75

Author(s):

Steve Watson

Keyword(s):

Platelet Activation ◽

Platelet Adhesion ◽

Collagen Fibers ◽

Severe Bleeding ◽

Platelet Surface ◽

Collagen Receptors ◽

Intracellular Signals ◽

Von Willebrand ◽

Surface Glycoproteins

IntroductionThe extracellular matrix protein, collagen, plays a primary role in hemostasis. Collagen fibers provide an important site for adhesion of platelets to the exposed subendothelium, trapping them at the site of vascular damage and enabling the formation of a monolayer of cells over the damaged area. Collagen fibers also stimulate platelet activation, leading to inside-out regulation of the integrin glycoprotein (GP) IIb-IIIa (also known as αIIbβ3), secretion from dense and α granules, generation of thromboxanes, and expression of procoagulant activity, all of which support the hemostatic process. The role of collagen in supporting platelet adhesion to the subendothelium is mediated through indirect and direct interactions. The indirect interaction is mediated through von Willebrand factor (vWF), which binds to the GP Ib-IX-V complex on the platelet surface.1-3 The interaction with vWF is critical for platelet adhesion at medium to high rates of flow because of the fast rate of association between vWF and GP Ib-IX. The importance of this interaction is demonstrated by the severe bleeding problems experienced by individuals with functional impairment of vWF (von Willebrand disease) or GP Ib-IX (Bernard-Soulier syndrome). At low rates of flow, collagen fibers are able to support adhesion in the absence of vWF through a direct interaction with a number of platelet surface glycoproteins i.e. collagen receptors,4,5 this also serves to support vWF-dependent adhesion at higher rates of flow by preventing dissociation. Crosslinking of platelet surface glycoproteins by collagen also generates intracellular signals, leading to platelet activation.The number of proteins on the platelet surface proposed to be collagen receptors is approaching double figures, but it is generally accepted that the integrin GP Ia-IIa (also known as α2β1) and glycoprotein VI (GP VI) are among the most important of these, playing critical roles in adhesion and activation, respectively6 (Fig. 1). This is illustrated by the mild bleeding problems of patients with a low level of expression or the presence of autoantibodies to GP Ia-IIa and the spontaneous, severe bleeding episodes that are occasionally seen in patients whose platelets are deficient in GP VI.6 There is evidence, however, that other collagen receptors have supporting roles in adhesion and activation. For example, GP VI supports platelet adhesion to collagen7 and GP IV, also known as CD36, may also play a similar role.8 The role of the recently cloned collagen receptor p65 in adhesion is not known. Evidence that the interaction of collagen with receptors, such as GPIV and p65, is of less importance than for interactions with GP Ia-IIa, and GP VI is provided by the absence of individuals with bleeding problems caused by deficiencies in these proteins. This is illustrated most clearly for GP IV, which is absent in 3% to 5 % of the Japanese population, and yet such individuals display no major vascular problems.Due to the large number of glycoproteins that bind collagen on the platelet surface, it has been difficult to gain a full understanding of the role of individual collagen receptors in adhesion and activation responses. This is complicated further by the interactions between vWF and GP Ib-IX-V, vWF or fibrinogen to activated GP IIb-IIIa especially as both glycoprotein receptors generate intracellular signals. The relative importance of individual collagen receptors in adhesion also varies with the rate of flow and between collagen types. A full discussion of platelet adhesion to collagen is beyond the scope of this article, and the reader is referred to a number of excellent recent reviews for further information.4-6,9,10 The present chapter focuses on the signaling events generated by the activation (or more correctly crosslinking) of platelet surface glycoproteins by collagen and the implications that this has for platelet activation under normal and diseased conditions.

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Fibrillar type I collagens enhance platelet-dependent thrombin generation via glycoprotein VI with direct support of α2β1 but not αIIbβ3 integrin

Thrombosis and Haemostasis ◽

10.1160/th04-12-0783 ◽

2005 ◽

Vol 94 (07) ◽

pp. 107-114 ◽

Cited By ~ 21

Author(s):

Christelle Lecut ◽

Martine Jandrot-Perrus ◽

Marion A. H. Feijge ◽

Judith M. E. M. Cosemans ◽

Johan W. M. Heemskerk

Keyword(s):

Thrombin Generation ◽

Type I Collagen ◽

Platelet Rich Plasma ◽

Procoagulant Activity ◽

Type I ◽

Platelet Surface ◽

Glycoprotein Vi ◽

Collagen Receptors ◽

Fibrillar Collagens

SummaryThe role of collagens and collagen receptors was investigated in stimulating platelet-dependent thrombin generation. Fibrillar type-I collagens, including collagen from human heart, were most potent in enhancing thrombin generation, in a way dependent on exposure of phosphatidylserine (PS) at the platelet surface. Soluble, non-fibrillar type-I collagen required pre-activation of integrin α2β1 with Mn2+ for enhancement of thrombin generation. With all preparations, blocking of glycoprotein VI (GPVI) with 9O12 antibody abrogated the collagen-enhanced thrombin generation, regardless of the α2β1 activation state. Blockade of α2β1 alone or antagonism of autocrine thromboxane A2 and ADP were less effective. Blockade of αIIbβ3 with abciximab suppressed thrombin generation in platelet-rich plasma, but this did not abolish the enhancing effect of collagens. The high activity of type-I fibrillar collagens in stimulating GPVI-dependent procoagulant activity was confirmed in whole-blood flow studies, showing that these collagens induced relatively high expression of PS. Together, these results indicate that: i) fibrillar type-I collagen greatly enhances thrombin generation, ii) GPVI-induced platelet activation is principally responsible for the procoagulant activity of fibrillar and non-fibrillar collagens, iii) α2β1 and signaling via autocrine mediators facilitate and amplify this GPVI activity, and iv) αIIbβ3 is not directly involved in the collagen effect.

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IκB kinase 2 is not essential for platelet activation

Blood Advances ◽

10.1182/bloodadvances.2019001044 ◽

2020 ◽

Vol 4 (4) ◽

pp. 638-643

Author(s):

Manuel Salzmann ◽

Sonja Bleichert ◽

Bernhard Moser ◽

Marion Mussbacher ◽

Mildred Haase ◽

...

Keyword(s):

Platelet Activation ◽

Active Site ◽

Bleeding Time ◽

Thrombus Formation ◽

Human Platelets ◽

Iκb Kinase ◽

Specific Deletion

Abstract Platelets are small anucleate cells that release a plethora of molecules to ensure functional hemostasis. It has been reported that IκB kinase 2 (IKK2), the central enzyme of the inflammatory NF-κB pathway, is involved in platelet activation, because megakaryocyte/platelet-specific deletion of exons 6 and 7 of IKK2 resulted in platelet degranulation defects and prolonged bleeding. We aimed to investigate the role of IKK2 in platelet physiology in more detail, using a platelet-specific IKK2 knockout via excision of exon 3, which makes up the active site of the enzyme. We verified the deletion on genomic and transcriptional levels in megakaryocytes and were not able to detect any residual IKK2 protein; however, platelets from these mice did not show any functional impairment in vivo or in vitro. Bleeding time and thrombus formation were not affected in platelet-specific IKK2-knockout mice. Moreover, platelet aggregation, glycoprotein GPIIb/IIIa activation, and degranulation were unaltered. These observations were confirmed by pharmacological inhibition of IKK2 with TPCA-1 and BMS-345541, which did not affect activation of murine or human platelets over a wide concentration range. Altogether, our results imply that IKK2 is not essential for platelet function.

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REDISTRIBUTION OF PLATELET GLYCOPROTEINS INDUCED BY ALLO- AND AUTOANTIBODIES

10.1055/s-0038-1644880 ◽

1987 ◽

Author(s):

S Santoso ◽

V Kiefel ◽

C Mueller-Eckhardt

Keyword(s):

Binding Sites ◽

Total Radioactivity ◽

Human Platelets ◽

Membrane Glycoproteins ◽

Platelet Surface ◽

In Vitro Effects ◽

Immunoblot Technique ◽

Antigen Antibody

It is now well established that two of the major membrane glycoproteins (GP) of human platelets, GP lb and Ilb/IIIa, are functionally prominent for adhesion, aggregation and carry the binding sites for allknown types of human platelet specific antibodies (ab). Although a number of in vitro effects of ab on platelet function have been described, the role of the GP specificity of the various ab with regard to membrane mobility and redistribution phenomena is asyet unknown.In this work, we studied the effect on platelet membrane redistribution of allo- ab, auto-aband a quinidine-dependent ab directed against various epitopes on GP lb, lib and Ilia using immunofluorescence and a quantitative radioimmunoassay. The platelet GP's carrying the corresponding epitopes were determined using immunoblot technique or radioimmuno-precipitation. When unfixed platelets were incubated with alio- or auto-ab against epitopes on GP liborGP IlIa cap formation and internalization of antigenantibody complexes were visualized by fluorescence. In contrast, no changes of antigen distribution were seen with auto-ab or quinidine- dependent ab directed against GP lb. To quantitate antigen-antibody complexes internalization a specially designed radioimmunoassay was employed. If unfixed platelets weretreated with allo- or auto-ab against GP lib or GP Ilia precipitous reduction of external radioactivity was found, whereas the total radioactivity remainedessentially unchanged. This indicated that a portionof approximately 50-70% of GP lib or GP Ilia had been removed from the platelet surface and had been internalized. Internalization could not be induced with auto-ab or quinidine dependent ab against GP lb.We conclude that membrane redistribution of human platelets can be induced by various human ab with specificity for GP lib and/or Ilia and is a function of the target GP rather than the source of therespective abSupported by Deutsche Forschungsgemeinschaft (Mu 277/9-6)

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