Long-term antithrombotic protection by in vivo depletion of platelet glycoprotein VI in mice.

Coronary artery thrombosis is often initiated by abrupt disruption of the atherosclerotic plaque and activation of platelets on the subendothelial layers in the disrupted plaque. The extracellular matrix protein collagen is the most thrombogenic constituent of the subendothelial layer; therefore, a selective inhibition of the collagen activation pathway in platelets may provide strong antithrombotic protection while preserving other platelet functions. Here we demonstrate that treatment of mice with a monoclonal antibody against the activating platelet collagen receptor glycoprotein VI (GPVI; JAQ1) results in specific depletion of the receptor from circulating platelets and abolished responses of these cells to collagen and collagen-related peptides (CRPs). JAQ1-treated mice were completely protected for at least 2 wk against lethal thromboembolism induced by infusion of a mixture of collagen (0.8 mg/kg) and epinephrine (60 microg/ml). The tail bleeding times in JAQ1-treated mice were only moderately increased compared with control mice probably because the treatment did not affect platelet activation by other agonists such as adenosine diphosphate or phorbol myristate acetate. These results suggest that GPVI might become a target for long-term prophylaxis of ischemic cardiovascular diseases and provide the first evidence that it is possible to specifically deplete an activating glycoprotein receptor from circulating platelets in vivo.

Negative regulators of platelet activation and adhesion.

Platelets are small anucleated cells that constantly patrol the cardiovascular system to preserve its integrity and prevent excessive blood loss where the vessel lining is breached. Their key challenge is to form a hemostatic plug under conditions of high shear forces. To do so, platelets have evolved a molecular machinery that enables them to sense trace amounts of signals at the site of damage and to rapidly shift from a non-adhesive to a pro-adhesive state. However, this highly efficient molecular machinery can also lead to unintended platelet activation and cause clinical complications such as thrombocytopenia and thrombosis. Thus, several checkpoints are in place to tightly control platelet activation and adhesiveness in space and time. In this review, we will discuss select negative regulators of platelet activation, which are critical to maintain patrolling platelets in a quiescent, non-adhesive state and/or to limit platelet adhesion to sites of injury.

Small molecule targeting the Rac1-NOX2 interaction prevents collagen-related peptide and thrombin-induced reactive oxygen species generation and platelet activation.

Essentials Reactive oxygen species (ROS) generation by NOX2 plays a critical role in platelet activation. Rac1 regulation of NOX2 is important for ROS generation. Small molecule inhibitor of the Rac1-p67phox interaction prevents platelet activation. Pharmacologic targeting of Rac1-NOX2 axis can be a viable approach for antithrombotic therapy. SUMMARY: Background Platelets from patients with X-linked chronic granulomatous disease or mice deficient in nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidase isoform NOX2 exhibit diminished reactive oxygen species (ROS) generation and platelet activation. Binding of Rac1 GTPase to p67phox plays a critical role in NOX2 activation by facilitating the assembly of the NOX2 enzyme complex. Objective We tested the hypothesis that Phox-I, a rationally designed small molecule inhibitor of Rac-p67phox interaction, may serve as an antithrombosis agent by suppressing ROS production and platelet activation. Results Collagen-related peptide (CRP) induced ROS generation in a time-dependent manner. Platelets from Rac1-/- mice or human platelets treated with NSC23766, a specific Rac inhibitor, produced significantly less ROS in response to CRP. Treatment of platelets with Phox-I inhibited diverse CRP-induced responses, including: (i) ROS generation; (ii) release of P-selectin; (iii) secretion of ATP; (iv) platelet aggregation; and (v) phosphorylation of Akt. Similarly, incubation of platelets with Phox-I inhibited thrombin-induced: (i) secretion of ATP; (ii) platelet aggregation; (iii) rise in cytosolic calcium; and (iv) phosphorylation of Akt. In mouse models, intraperitoneal administration of Phox-I inhibited: (i) collagen-induced platelet aggregation without affecting the tail bleeding time and (ii) in vivo platelet adhesion/accumulation at the laser injury sites on the saphenous vein without affecting the time for complete cessation of blood loss. Conclusions Small molecule targeting of the Rac1-p67phox interaction may present an antithrombosis regimen by preventing GPVI- and non-GPVI-mediated NOX2 activation, ROS generation and platelet function without affecting the bleeding time.

Platelet immunoreceptor tyrosine-based activation motif (ITAM) and hemITAM signaling and vascular integrity in inflammation and development.

Platelets are essential for maintaining hemostasis following mechanical injury to the vasculature. Besides this established function, novel roles of platelets are becoming increasingly recognized, which are critical in non-injury settings to maintain vascular barrier integrity. For example, during embryogenesis platelets act to support the proper separation of blood and lymphatic vessels. This role continues beyond birth, where platelets prevent leakage of blood into the lymphatic vessel network. During the course of inflammation, platelets are necessary to prevent local hemorrhage due to neutrophil diapedesis and disruption of endothelial cell-cell junctions. Surprisingly, platelets also work to secure tumor-associated blood vessels, inhibiting excessive vessel permeability and intra-tumor hemorrhaging. Interestingly, many of these novel platelet functions depend on immunoreceptor tyrosine-based activation motif (ITAM) signaling but not on signaling via G protein-coupled receptors, which plays a crucial role in platelet plug formation at sites of mechanical injury. Murine platelets express two ITAM-containing receptors: the Fc receptor γ-chain (FcRγ), which functionally associates with the collagen receptor GPVI, and the C-type lectin-like 2 (CLEC-2) receptor, a hemITAM receptor for the mucin-type glycoprotein podoplanin. Human platelets express an additional ITAM receptor, FcγRIIA. These receptors share common downstream effectors, including Syk, SLP-76 and PLCγ2. Here we will review the recent literature that highlights a critical role for platelet GPVI/FcRγ and CLEC-2 in vascular integrity during development and inflammation in mice and discuss the relevance to human disease.

Tissue factor-positive tumor microvesicles activate platelets and enhance thrombosis in mice.

UNLABELLED: ESSENTIALS: Cancer patients have a high rate of venous thrombosis (VT) but the underlying mechanisms are unknown. Tumor-derived, tissue factor-positive microvesicles in platelet activation in vitro and in vivo were studied. Tumor-derived, tissue factor-positive microvesicles enhanced VT in mice. Platelets may contribute to VT in some cancer patients, and this could be prevented with antiplatelet drugs. BACKGROUND: Cancer patients have an approximately 4-fold increased risk of venous thromboembolism (VTE) compared with the general population, and cancer patients with VTE have reduced survival. Tumor cells constitutively release small membrane vesicles called microvesicles (MVs) that may contribute to thrombosis in cancer patients. Clinical studies have shown that levels of circulating tumor-derived, tissue factor-positive (TF(+) ) MVs in pancreatic cancer patients are associated with VTE. Objectives We tested the hypothesis that TF(+) tumor-derived MVs (TMVs) activate platelets in vitro and in mice. MATERIALS AND METHODS: We selected two human pancreatic adenocarcinoma cell lines expressing high (BxPc-3) and low (L3.6pl) levels of TF as models to study the effect of TF(+) TMVs on platelets and thrombosis. RESULTS AND CONCLUSIONS: We found that both types of TF(+) TMVs activated human platelets and induced aggregation in vitro in a TF and thrombin-dependent manner. Further, injection of BxPc-3 TF(+) TMVs triggered platelet activation in vivo and enhanced thrombosis in two mouse models of venous thrombosis in a TF-dependent manner. Importantly, BxPc-3 TF(+) TMV-enhanced thrombosis was reduced in Par4-deficient mice and in wild-type mice treated with clopidogrel, suggesting that platelet activation was required for enhanced thrombosis. These studies suggest that TF(+) TMV-induced platelet activation contributes to thrombosis in cancer patients.

OC-04 - Tissue factor positive microvesicles activate platelets in vitro and in vivo and enhance thrombosis in mice.

INTRODUCTION: Cancer patients have a 4- to 7- fold increased risk of venous thromboembolism (VTE) compared with general population. Most tumor cells express tissue factor (TF) and constitutively release small membrane microvesicles called tumor microvesicles (TMVs). Clinical studies have shown that circulating MP-TF activity is associated with VTE in pancreatic cancer but not in other types of cancer. Thrombin is a potent platelet agonist and activates platelets via protease activated receptors (PARs). AIM: To determine the contribution of the TF+ TMV-thrombin-platelet pathway to cancer-associated thrombosis. MATERIALS AND METHODS: A human pancreatic adenocarcinoma cell line expressing high levels of TF (BxPc-3) was selected to study the effect of TF+ TMVs on platelet activation and thrombosis. RESULTS: TF+ TMVs induced platelet activation in vitro in a thrombin-dependent manner. The presence of orthotopically grown BxPc-3 tumors in mice was associated with increased levels of thrombin-antithrombin III complexes (TATc) and larger thrombi in an inferior vena cava stenosis model compared with control mice. Furthermore, injection of BxPc-3 TF+ TMVs into mice triggered platelet activation and enhanced venous thrombosis in a TF-dependent manner. Importantly, BxPc-3 TF+ TMV-enhanced thrombosis was reduced in Par4-deficient mice and wild-type mice treated with the platelet inhibitor clopidogrel, suggesting that platelet activation was required for the enhanced thrombosis. CONCLUSIONS: These studies suggest that platelet inhibitors may reduce thrombosis in cancer patients with elevated levels of TF+ TMVs.

Novel mouse hemostasis model for real-time determination of bleeding time and hemostatic plug composition.

INTRODUCTION: Hemostasis is a rapid response by the body to stop bleeding at sites of vessel injury. Both platelets and fibrin are important for the formation of a hemostatic plug. Mice have been used to uncover the molecular mechanisms that regulate the activation of platelets and coagulation under physiologic conditions. However, measurements of hemostasis in mice are quite variable, and current methods do not quantify platelet adhesion or fibrin formation at the site of injury. METHODS: We describe a novel hemostasis model that uses intravital fluorescence microscopy to quantify platelet adhesion, fibrin formation and time to hemostatic plug formation in real time. Repeated vessel injuries of ~ 50-100 µm in diameter were induced with laser ablation technology in the saphenous vein of mice. RESULTS: Hemostasis in this model was strongly impaired in mice deficient in glycoprotein Ibα or talin-1, which are important regulators of platelet adhesiveness. In contrast, the time to hemostatic plug formation was only minimally affected in mice deficient in the extrinsic tissue factor (TF(low)) or the intrinsic factor IX coagulation pathways, even though platelet adhesion was significantly reduced. A partial reduction in platelet adhesiveness obtained with clopidogrel led to instability within the hemostatic plug, especially when combined with impaired coagulation in TF(low) mice. CONCLUSIONS: In summary, we present a novel, highly sensitive method to quantify hemostatic plug formation in mice. On the basis of its sensitivity to platelet adhesion defects and its real-time imaging capability, we propose this model as an ideal tool with which to study the efficacy and safety of antiplatelet agents.

The P2Y(12) receptor induces platelet aggregation through weak activation of the alpha(IIb)beta(3) integrin--a phosphoinositide 3-kinase-dependent mechanism.

High concentrations of adenosine-5'-diphosphate ADP are able to induce partial aggregation without shape change of P2Y(1) receptor-deficient mouse platelets through activation of the P2Y(12) receptor. In the present work we studied the transduction pathways selectively involved in this phenomenon. Flow cytometric analyses using R-phycoerythrin-conjugated JON/A antibody (JON/A-PE), an antibody which recognizes activated mouse alpha(IIb)beta(3) integrin, revealed a low level activation of alpha(IIb)beta(3) in P2Y(1) receptor-deficient platelets in response to 100 microM ADP or 1 microM 2MeS-ADP. Adrenaline induced no such activation but strongly potentiated the effect of ADP in a dose-dependent manner. Global phosphorylation of (32)P-labeled platelets showed that P2Y(12)-mediated aggregation was not accompanied by an increase in the phosphorylation of myosin light chain (P(20)) or pleckstrin (P(47)) and was not affected by the protein kinase C (PKC) inhibitor staurosporine. On the other hand, two unrelated phosphoinositide 3-kinase inhibitors, wortmannin and LY294002, inhibited this aggregation. Our results indicate that (i) the P2Y(12) receptor is able to trigger a P2Y(1) receptor-independent inside-out signal leading to alpha(IIb)beta(3) integrin activation and platelet aggregation, (ii) ADP and adrenaline use different signaling pathways which synergize to activate the alpha(IIb)beta(3) integrin, and (iii) the transduction pathway triggered by the P2Y(12) receptor is independent of PKC but dependent on phosphoinositide 3-kinase.

CalDAG-GEFI deficiency protects mice from FcγRIIa-mediated thrombotic thrombocytopenia induced by CD40L and ß2GPI immune complexes.

INTRODUCTION: Platelet activation via the Fcγ receptor IIa (FcγRIIa) is implicated in the pathogenesis of immune complex (IC)-mediated thrombocytopenia and thrombosis (ITT). We previously showed that ICs composed of antigen and antibodies targeting CD40 ligand (CD40L) or ß2 Glycoprotein I (ß2GPI) induce ITT in mice transgenic for human FcγRIIa (hFcR) but not wild-type controls (which lack FcγRIIa). Here we evaluated the contribution of the guanine nucleotide exchange factor, CalDAG-GEFI, and P2Y12, key regulators of Rap1 signaling in platelets, to ITT induced by these clinically relevant ICs. METHODS: Pre-formed anti-CD40L or anti-ß2GPI ICs were injected into hFcR/Caldaggef1(+/+) or hFcR/Caldaggef1(-/-) mice, with or without clopidogrel pretreatment. Animals were observed for symptoms of shock for 30 min, during which time core body temperature was monitored. Platelet counts were obtained before and 30 min after IC injection. Lungs were assessed for thrombosis by histology or near-infrared imaging. RESULTS: Both CD40L and ß2GPI ICs rapidly induced severe thrombocytopenia, shock and a reduction in body temperature in hFcR/Caldaggef1(+/+) mice. hFcR/Caldaggef1(-/-) mice were protected from CD40L and ß2GPI IC-induced thrombocytopenia and shock, whereas P2Y12 inhibition had only a modest effect on IC-induced ITT. Consistent with these findings, IC-induced integrin activation in vitro and the accumulation of activated platelets in the lungs of IC-challenged mice was strongly dependent on CalDAG-GEFI. CONCLUSIONS: Our studies demonstrate that CalDAG-GEFI plays a critical role in platelet activation, thrombocytopenia and thrombosis induced by clinically relevant ICs in mice. Thus, CalDAG-GEFI may be a promising target for the intervention of IC-associated, FcγRIIa-mediated thrombotic conditions.

The fibrinogen γA/γ' isoform does not promote acute arterial thrombosis in mice.

BACKGROUND: Elevated plasma fibrinogen is associated with arterial thrombosis in humans and promotes thrombosis in mice by increasing fibrin formation and thrombus fibrin content. Fibrinogen is composed of six polypeptide chains: (Aα, Bß, and γ)2. Alternative splicing of the γ chain leads to a dominant form (γA/γA) and a minor species (γA/γ'). Epidemiological studies have detected elevated γA/γ' fibrinogen in patients with arterial thrombosis, suggesting that this isoform promotes thrombosis. However, in vitro data show that γA/γ' is anticoagulant due to its ability to sequester thrombin and suggest its expression is upregulated in response to inflammatory processes. OBJECTIVE: To determine whether γA/γ' fibrinogen is prothrombotic in vivo. METHODS: We separated γA/γA and γA/γ' fibrinogen from human plasma-purified fibrinogen and determined the effects on in vitro plasma clot formation and on in vivo thrombus formation and circulating thrombin-antithrombin complexes in mice. RESULTS AND CONCLUSIONS: Both γA/γA and γA/γ' fibrinogen were cleaved by murine and human thrombin and were incorporated into murine and human clots. When γA/γA or γA/γ' was spiked into plasma, γA/γA increased the fibrin formation rate to a greater extent than γA/γ'. In mice, compared to controls, γA/γA infusion shortened the time to carotid artery occlusion, whereas γA/γ' infusion did not. Additionally, γA/γ' infusion led to lower levels of plasma thrombin-antithrombin complexes following arterial injury, whereas γA/γA infusion did not. These data suggest that γA/γ' binds thrombin in vivo and decreases prothrombotic activity. Together, these findings indicate that elevated levels of γA/γA fibrinogen promote arterial thrombosis in vivo, whereas γA/γ' does not.

Relative contributions of stromal interaction molecule 1 and CalDAG-GEFI to calcium-dependent platelet activation and thrombosis.

BACKGROUND: Stromal interaction molecule 1 (STIM1) was recently identified as a critical component of store-operated calcium entry (SOCE) in platelets. We previously reported the Ca(2+) -sensing guanine nucleotide exchange factor CalDAG-GEFI as a critical molecule in Ca(2+) signaling in platelets. OBJECTIVE: To evaluate the contribution of STIM1/SOCE to Ca(2+) -dependent platelet activation and thrombosis, we here compared the activation responses of platelets lacking STIM1 and platelets lacking CalDAG-GEFI. METHODS: The murine Stim1 gene was conditionally deleted in the megakaryocyte/platelet lineage. CalDAG-GEFI(-/-) and Stim1(fl/fl) PF4-Cre mice, along with littermate control mice, were used for in vitro and in vivo experiments under flow as well as static conditions. RESULTS: Integrin α(IIb) ß(3) -mediated aggregation was markedly impaired in CalDAG-GEFI-deficient but not STIM1-deficient platelets, under both static and flow conditions. In contrast, deficiency in either STIM1 or CalDAG-GEFI significantly impaired the ability of platelets to express phosphatidylserine on the cell surface. When subjected to a laser injury thrombosis model, mice lacking STIM1 in platelets were characterized by the formation of unstable platelet-rich thrombi and delayed and reduced fibrin generation in injured arterioles. In CalDAG-GEFI(-/-) mice, fibrin generation was also delayed and reduced, but platelet accumulation was almost abolished. CONCLUSIONS: Our studies suggest that: (i) STIM1/SOCE is critical for the procoagulant activity but not the proadhesive function of platelets; and (ii) at the site of vascular injury, STIM1 and CalDAG-GEFI are critical for the first wave of thrombin generation mediated by procoagulant platelets.

Novel molecules in calcium signaling in platelets.

A rise in the intracellular calcium (Ca2+) concentration is a major component of the signaling mechanisms regulating platelet function in thrombosis and hemostasis. Previous studies, however, failed to identify many key molecules regulating Ca2+ signaling in platelets. Here, we review recent findings, which identified CalDAG-GEFI as a critical Ca2+ sensor that links increases in intracellular Ca2+ to integrin activation, TxA2 formation, and granule release in stimulated platelets. Furthermore, we summarize work that lead to the discovery of STIM1 and Orai1 as key regulators of store-operated calcium entry (SOCE) in platelets. A short discussion on the usefulness of each molecule as a potential new target for antiplatelet therapy is included.

Identification of critical antigen-specific mechanisms in the development of immune thrombocytopenic purpura in mice.

The pathogenic effects of antiplatelet antibodies were investigated in mice. Monoclonal antibodies (mAbs) of different immunoglobulin G subclass directed against mouse GPIIbIIIa, GPIIIa, GPIbalpha, GPIb-IX, GPV, and CD31 were generated and characterized biochemically. MAbs against GPIb-IX, GPV, CD31, and linear epitopes on GPIIIa had mild and transient effects on platelet counts and induced no spontaneous bleeding. Anti-GPIbalpha mAbs induced profound irreversible thrombocytopenia (< 3% of normal) by Fc-independent mechanisms but only had minor effects on hematocrits. In contrast, injection of intact mAbs, but not F(ab)(2) fragments, against conformational epitopes on GPIIbIIIa, induced irreversible thrombocytopenia, acute systemic reactions, hypothermia, decreased hematocrits, and a paradoxical loss of surface GPIIbIIIa on platelets in vivo, the latter suggesting the formation of platelet-derived microparticles. Blockage of platelet-activating factor receptors inhibited the acute reactions, but not thrombocytopenia, loss of GPIIbIIIa, and decreases in hematocrits. Repeated injections of low doses of anti-GPIIbIIIa antibodies resulted in profound thrombocytopenia and bleeding, whereas no acute systemic reactions were observed. These data strongly suggest that the identity of the target antigen recognized by antiplatelet antibodies determines the mechanisms of platelet destruction and the severity of bleeding in mice, the latter depending on previously unrecognized anti-GPIIbIIIa-specific inflammatory mechanisms.

Evidence for two distinct epitopes within collagen for activation of murine platelets.

It has recently been shown that the monoclonal antibody JAQ1 to murine glycoprotein VI (GPVI) can cause aggregation of mouse platelets upon antibody cross-linking and that collagen-induced platelet aggregation can be inhibited by preincubation of platelets with JAQ1 in the absence of cross-linking (Nieswandt, B., Bergmeier, W., Schulte, V., Rackebrandt, K., Gessner, J. E., and Zirngibl, H. (2000) J. Biol. Chem. 275, 23998-24002). In the present study, we have shown that cross-linking of GPVI by JAQ1 results in tyrosine phosphorylation of the same profile of proteins as that induced by collagen, including the Fc receptor (FcR) gamma-chain, Syk, LAT, SLP-76, and phospholipase C gamma 2. In contrast, platelet aggregation and tyrosine phosphorylation of these proteins were inhibited when mouse platelets were preincubated with JAQ1 in the absence of cross-linking and were subsequently stimulated with a collagen-related peptide (CRP) that is specific for GPVI and low concentrations of collagen. However, at higher concentrations of collagen, but not CRP, aggregation of platelets and tyrosine phosphorylation of the above proteins (except for the adapter LAT) is re-established despite the presence of JAQ1. These observations suggest that a second activatory binding site, which is distinct from the CRP binding site on GPVI on mouse platelets, is occupied in the presence of high concentrations of collagen. Although this could be a second site on GPVI that is activated by a novel motif within the collagen molecule, the absence of LAT phosphorylation in response to collagen in the presence of JAQ1 suggests that this is more likely to be caused by activation of a second receptor that is also coupled to the FcR gamma-chain. The possibility that this response is mediated by a receptor that is not coupled to FcR gamma-chain is excluded on the grounds that aggregation is absent in platelets from FcR gamma-chain-deficient mice.

Expression and function of the mouse collagen receptor glycoprotein VI is strictly dependent on its association with the FcRgamma chain.

Platelet glycoprotein (GP) VI has been proposed as the major collagen receptor for activation of human platelets. Human GPVI belongs to the immunoglobulin superfamily and is noncovalently associated with the FcRgamma chain that is involved in signaling through the receptor. In mice, similar mechanisms seem to exist as platelets from FcRgamma chain-deficient mice do not aggregate in response to collagen. However, the activating collagen receptor on mouse platelets has not been definitively identified. In the current study we examined the function and in vivo expression of GPVI in control and FcRgamma chain-deficient mice with the first monoclonal antibody against GPVI (JAQ1). On wild type platelets, JAQ1 inhibited platelet aggregation induced by collagen but not PMA or thrombin. Cross-linking of bound JAQ1, on the other hand, induced aggregation of wild type but not FcRgamma chain-deficient platelets. JAQ1 stained platelets and megakaryocytes from wild type but not FcRgamma chain-deficient mice. Furthermore, JAQ1 recognized GPVI (approximately 60 kDa) in immunoprecipitation and Western blot experiments with wild type but not FcRgamma chain-deficient platelets. These results strongly suggest that GPVI is the collagen receptor responsible for platelet activation in mice and demonstrate that the association with the FcRgamma chain is critical for its expression and function.

Structural and functional characterization of the mouse von Willebrand factor receptor GPIb-IX with novel monoclonal antibodies.

Five novel monoclonal antibodies (mAbs; p0p 1-5) were used to characterize the structural and functional properties and the in vivo expression of the murine GPIb-IX complex (von Willebrand factor receptor). The molecular weights of the subunits are similar to the human homologs: GPIbalpha (150 kd), GPIbbeta (25 kd), and GPIX (25 kd). Activation of platelets with thrombin or PMA predominantly induced shedding of glycocalicin (GC; 130 kd) but only low levels of receptor internalization. The GC concentration in normal mouse plasma was found to be at least 10 times higher than that described for human plasma (approximately 25 microg/mL versus 1-2 microg/mL). Two additional cleavage sites for unidentified platelet-derived proteases were found on GPIbalpha, as demonstrated by the generation of 3 N-terminal fragments during in vitro incubation of washed platelets (GC, 60 kd, 45 kd). Occupancy of GPIbalpha with p0p mAbs or F(ab)(2)-fragments resulted in aggregate formation in vitro and rapid irreversible thrombocytopenia in vivo, irrespective of the exact binding epitopes of the individual antibodies. GPIb-IX was not detectable immunohistochemically on endothelial cells in the major organs under normal or inflammatory conditions. The authors conclude that the mouse system might become an interesting model for studies on GPIb-IX function and regulation.

Rhodocytin (aggretin) activates platelets lacking alpha(2)beta(1) integrin, glycoprotein VI, and the ligand-binding domain of glycoprotein Ibalpha.

Although alpha(2)beta(1) integrin (glycoprotein Ia/IIa) has been established as a platelet collagen receptor, its role in collagen-induced platelet activation has been controversial. Recently, it has been demonstrated that rhodocytin (also termed aggretin), a snake venom toxin purified from the venom of Calloselasma rhodostoma, induces platelet activation that can be blocked by monoclonal antibodies against alpha(2)beta(1) integrin. This finding suggested that clustering of alpha(2)beta(1) integrin by rhodocytin is sufficient to induce platelet activation and led to the hypothesis that collagen may activate platelets by a similar mechanism. In contrast to these findings, we provided evidence that rhodocytin does not bind to alpha(2)beta(1) integrin. Here we show that the Cre/loxP-mediated loss of beta(1) integrin on mouse platelets has no effect on rhodocytin-induced platelet activation, excluding an essential role of alpha(2)beta(1) integrin in this process. Furthermore, proteolytic cleavage of the 45-kDa N-terminal domain of glycoprotein (GP) Ibalpha either on normal or on beta(1)-null platelets had no significant effect on rhodocytin-induced platelet activation. Moreover, mouse platelets lacking both alpha(2)beta(1) integrin and the activating collagen receptor GPVI responded normally to rhodocytin. Finally, even after additional proteolytic removal of the 45-kDa N-terminal domain of GPIbalpha rhodocytin induced aggregation of these platelets. These results demonstrate that rhodocytin induces platelet activation by mechanisms that are fundamentally different from those induced by collagen.

Evidence for cross-talk between glycoprotein VI and Gi-coupled receptors during collagen-induced platelet aggregation.

Collagen-induced platelet aggregation is a complex process and involves synergistic action of integrins, immunoglobulin (Ig)-like receptors, G-protein-coupled receptors and their ligands, most importantly collagen itself, thromboxane A(2) (TXA(2)), and adenosine diphosphate (ADP). The precise role of each of these receptor systems in the overall processes of activation and aggregation, however, is still poorly defined. Among the collagen receptors expressed on platelets, glycoprotein (GP) VI has been identified to play a crucial role in collagen-induced activation. GPVI is associated with the FcRgamma chain, which serves as the signal transducing unit of the receptor complex. It is well known that clustering of GPVI by highly specific agonists results in platelet activation and irreversible aggregation, but it is unclear whether collagen has the same effect on the receptor. This study shows that platelets from Galphaq-deficient mice, despite their severely impaired response to collagen, normally aggregate on clustering of GPVI, suggesting this not to be the principal mechanism by which collagen activates platelets. On the other hand, dimerization of GPVI by a monoclonal antibody (JAQ1), which by itself did not induce aggregation, provided a sufficient stimulus to potentiate platelet responses to Gi-coupled, but not Gq-coupled, agonists. The combination of JAQ1 and adrenaline or ADP, but not serotonin, resulted in alpha(IIb)beta(3)-dependent aggregation that occurred without intracellular calcium mobilization and shape change in the absence of Galphaq or the P2Y(1) receptor. Together, these results provide evidence for a cross-talk between (dimerized) GPVI and Gi-coupled receptors during collagen-induced platelet aggregation. (Blood. 2001;97:3829-3835)

Glycoprotein VI but not alpha2beta1 integrin is essential for platelet interaction with collagen.

Platelet adhesion on and activation by components of the extracellular matrix are crucial to arrest post-traumatic bleeding, but can also harm tissue by occluding diseased vessels. Integrin alpha2beta1 is thought to be essential for platelet adhesion to subendothelial collagens, facilitating subsequent interactions with the activating platelet collagen receptor, glycoprotein VI (GPVI). Here we show that Cre/loxP-mediated loss of beta1 integrin on platelets has no significant effect on the bleeding time in mice. Aggregation of beta1-null platelets to native fibrillar collagen is delayed, but not reduced, whereas aggregation to enzymatically digested soluble collagen is abolished. Furthermore, beta1-null platelets adhere to fibrillar, but not soluble collagen under static as well as low (150 s(-1)) and high (1000 s(-1)) shear flow conditions, probably through binding of alphaIIbbeta3 to von Willebrand factor. On the other hand, we show that platelets lacking GPVI can not activate integrins and consequently fail to adhere to and aggregate on fibrillar as well as soluble collagen. These data show that GPVI plays the central role in platelet-collagen interactions by activating different adhesive receptors, including alpha2beta1 integrin, which strengthens adhesion without being essential.