B Nieswandt

Publications of B Nieswandt

• Defective diacylglycerol-induced Ca(2+) entry but normal agonist-induced activation responses in TRPC6-deficient mouse platelets.

  Authors: G Ramanathan, S Gupta, I Thielmann, I Pleines, D Varga-Szabo, F May, C Mannhalter, A Dietrich, B Nieswandt, A Braun

  Journal of thrombosis and haemostasis : JTH. 12/2011; 10(3):419-29.

  Summary.  Background: Platelet adhesion, activation and aggregation at sites of vascular injury are essential processes for primary hemostasis. Elevation of the intracellular Ca(2+) concentration isSummary.  Background: Platelet adhesion, activation and aggregation at sites of vascular injury are essential processes for primary hemostasis. Elevation of the intracellular Ca(2+) concentration is a central event in platelet activation but the underlying mechanisms are not fully understood. Store-operated calcium entry (SOCE) through Orai1 was shown to be the main Ca(2+) influx pathway in murine platelets, but there are additional non-store-operated Ca(2+) (non-SOC) and receptor operated Ca(2+) (ROC) channels expressed in the platelet plasma membrane.Objective: Canonical transient receptor potential (TRPC) channel 6 is found both in human and murine platelets and has been proposed to mediate diacylglycerol (DAG) activated ROCE but also a role in the regulation of SOCE has been suggested.Methods: To investigate the function of TRPC6 in platelet Ca(2+) signaling and activation, we analyzed platelets from mice deficient in TRPC6 using a wide range of in vitro and in vivo assays.Results: In the mutant platelets, DAG activated Ca(2+) influx was found to be abolished. However, this did not significantly affect SOCE or agonist induced Ca(2+) responses. Platelet function in vitro and in vivo was also unaltered in the absence of TRPC6.Conclusion: Our results indicate that DAG activated ROCE is mediated exclusively by TRPC6 in murine platelets, but this Ca(2+) influx has no major functional relevance for hemostasis and thrombosis. Further, in contrast to previous suggestions, based on studies with human platelets, TRPC6 appears to play an insignificant role in the regulation of SOCE in murine platelets.

• [Acute ischemic stroke : New approaches to antithrombotic treatment.]

  Authors: P Kraft, B Nieswandt, G Stoll, C Kleinschnitz

  Der Nervenarzt. 09/2011;

  The only recommended therapy in the acute phase of ischemic stroke is thrombolysis within 4.5-(6) h after symptom onset. For secondary stroke prevention platelet inhibitors or, in cases of cardiacThe only recommended therapy in the acute phase of ischemic stroke is thrombolysis within 4.5-(6) h after symptom onset. For secondary stroke prevention platelet inhibitors or, in cases of cardiac embolism, anticoagulants are used. However, these substances bear significant limitations: either they show only moderate efficacy (platelet inhibitors), or they are associated with a considerable bleeding risk (rt-PA, anticoagulants). Although the majority of strokes are caused by embolic or thrombotic vessel occlusion, strikingly little is known about the pathophysiological role of platelets and their local function in the brain vasculature. The recent development of novel transgenic mouse lines paved the way for the in-depth analysis of the different molecular steps of thrombus formation involving platelets and the plasma coagulation cascade in models of acute ischemic stroke. It was demonstrated that prevention of early platelet adhesion to the damaged vessel wall by blocking the platelet surface receptors GPIbα or GPVI dramatically protects against experimental stroke without increasing the frequency of intracranial hemorrhage. Moreover, the critical involvement of the blood coagulation factor XII (FXII)-driven intrinsic coagulation cascade in thrombus formation during the course of ischemic brain damage could be unraveled thereby disproving established concepts of hemostasis. Based on these findings novel pharmacological blockers of GPIbα and FXIIa were designed that likewise proved to be safe and effective in animal stroke studies. Those compounds now lay the groundwork for a novel and intriguing concept in ischemic stroke and other thromboembolic diseases: antithrombosis devoid of any bleeding complications. Further preclinical testing is currently ongoing.

• Platelet adhesion and activation mechanisms in arterial thrombosis and ischaemic stroke.

  Authors: B Nieswandt, I Pleines, M Bender

  Journal of thrombosis and haemostasis : JTH. 07/2011; 9 Suppl 1:92-104.

  Platelet adhesion, activation and aggregation on the exposed subendothelial extracellular matrix (ECM) are essential for haemostasis, but may also lead to occlusion of diseased vessels. Binding ofPlatelet adhesion, activation and aggregation on the exposed subendothelial extracellular matrix (ECM) are essential for haemostasis, but may also lead to occlusion of diseased vessels. Binding of the glycoprotein (GP)Ib-V-IX complex to immobilised von Willebrand factor (VWF) initiates adhesion of flowing platelets to the ECM, and thereby enables the collagen receptor GPVI to interact with its ligand and to mediate platelet activation. This process is reinforced by locally produced thrombin and platelet-derived secondary mediators, such as adenosine diphosphate (ADP) and thromboxane A(2) (TxA(2)). Together, these events promote a shift of β1 and β3 integrins from a low to a high affinity state for their ligands through 'inside-out' signalling allowing firm platelet adhesion and aggregation. Formed platelet aggregates are stabilised by fibrin formation and signalling events between adjacent platelets involving multiple platelet receptors, such as the newly discovered C-type lectin-like receptor 2 (CLEC-2). While occlusive thrombus formation is the principal pathogenic event in myocardial infarction, the situation is more complex in ischaemic stroke where infarct development often progresses despite sustained early reperfusion of previously occluded major intracranial arteries, a process referred to as 'reperfusion injury'. Increasing experimental evidence now suggests that early platelet adhesion and activation events, orchestrate a 'thrombo-inflammatory' cascade in this setting, whereas platelet aggregation and thrombus formation are not required. This review summarises recent developments in understanding the principal platelet adhesion receptor systems with a focus on their involvement in arterial thrombosis and ischaemic stroke models.

• Genetic and antibody-induced glycoprotein VI deficiency equally protects mice from mechanically and FeCl(3) -induced thrombosis.

  Authors: M Bender, I Hagedorn, B Nieswandt

  Journal of thrombosis and haemostasis : JTH. 05/2011; 9(7):1423-6.

• EMMPRIN (CD147/basigin) mediates platelet-monocyte interactions in vivo and augments monocyte recruitment to the vascular wall.

  Authors: C Schulz, M-L von Brühl, V Barocke, P Cullen, K Mayer, R Okrojek, A Steinhart, Z Ahmad, E Kremmer, B Nieswandt, J Frampton, S Massberg, R Schmidt

  Journal of thrombosis and haemostasis : JTH. 02/2011; 9(5):1007-19.

  Platelets play a central role in hemostasis, in inflammatory diseases such as atherosclerosis, and during thrombus formation following vascular injury. Thereby, platelets interact intensively withPlatelets play a central role in hemostasis, in inflammatory diseases such as atherosclerosis, and during thrombus formation following vascular injury. Thereby, platelets interact intensively with monocytes and enhance their recruitment to the vascular wall. To investigate the role of the extracellular matrix metalloproteinase inducer (EMMPRIN) in platelet-monocyte interactions. Isolated human monocytes were perfused in vitro over firmly adherent platelets to allow investigation of the role of EMMPRIN in platelet-monocyte interactions under flow conditions. Monocytes readily bound to surface-adherent platelets. Both antibody blockade and gene silencing of monocyte EMMPRIN substantially attenuated firm adhesion of monocytes to platelets at arterial and venous shear rates. In vivo, platelet interactions with the murine monocyte cell line ANA-1 were significantly decreased when ANA-1 cells were pretreated with EMMPRIN-silencing small interfering RNA prior to injection into wild-type mice. Using intravital microscopy, we showed that recruitment of EMMPRIN-silenced ANA-1 to the injured carotid artery was significantly reduced as compared with control cells. Further silencing of EMMPRIN resulted in significantly fewer ANA-1-platelet aggregates in the mouse circulation as determined by flow cytometry. Finally, we identified glycoprotein (GP)VI as a critical corresponding receptor on platelets that mediates interaction with monocyte EMMPRIN. Thus, blocking of GPVI inhibited the effect of EMMPRIN on firm monocyte adhesion to platelets under arterial flow conditions in vitro, and abrogated EMMPRIN-mediated platelet-monocyte aggregate formation in vivo. EMMPRIN supports platelet-monocyte interactions and promotes monocyte recruitment to the arterial wall. Therefore, EMMPRIN might represent a novel target to reduce vascular inflammation and atherosclerotic lesion development.

• Arterial thrombus formation. Novel mechanisms and targets Novel mechanisms and targets.

  Authors: I Hagedorn, T Vögtle, B Nieswandt

  Hämostaseologie. 08/2010; 30(3):127-35.

  Platelet and coagulation factor-dependent thrombus formation is critical to limit posttraumatic blood loss at sites of vascular injury. However, under pathological conditions like rupture of anPlatelet and coagulation factor-dependent thrombus formation is critical to limit posttraumatic blood loss at sites of vascular injury. However, under pathological conditions like rupture of an atherosclerotic plaque, it may also lead to vessel occlusion causing myocardial infarction or stroke. Therefore, antithrombotic treatment is the prime therapeutic option in the prophylaxis and treatment of ischaemic cardio- and cerebrovascular diseases. The use of existing antithrombotic agents is, however, limited by their inherent effect on primary haemostasis. In recent years, major advances have been made in understanding the mechanisms of thrombus formation in haemostasis and thrombosis and some studies raised the interesting possibility that occlusive thrombus formation and haemostasis may involve partially different mechanisms. This review briefly summarizes these developments and highlights newly identified mechanisms involved in platelet adhesion and activation, intracellular calcium signaling, integrin activation and initiation of coagulation. The suitability of these pathways as novel targets for antithrombotic therapy is discussed.

• The role of glycoprotein Ibalpha and von Willebrand factor interaction in stroke development.

  Authors: G Stoll, C Kleinschnitz, B Nieswandt

  Hämostaseologie. 08/2010; 30(3):136-8.

  Ischaemic stroke is a devastating disease with limited treatment options due to numerous uncertainties regarding the underlying pathophysiology. The contribution of glycoprotein (GP)Ibalpha and vonIschaemic stroke is a devastating disease with limited treatment options due to numerous uncertainties regarding the underlying pathophysiology. The contribution of glycoprotein (GP)Ibalpha and von Willebrand factor (VWF) in stroke development has only recently been established in mice. Complete blockade of GPIbalpha led to a significant reduction of infarct volumes in mice undergoing one hour of transient middle cerebral artery occlusion (tMCAO). High shear-induced changes in VWF confirmation are a prerequisite for VWF binding to collagen and GPIbalpha expressed on platelets. Importantly, transgenic VWF-/- mice were similarly protected against ischemic stroke after tMCAO, and hydrodynamic injection of a VWF-encoding plasmid restored VWF serum levels and the susceptibility towards stroke. Secreted VWF is rapidly cleaved by ADAMTS13. Accordingly, ADAMTS13 deficient mice developed larger infarction after tMCAO, while infusion of recombinant ADAMTS13 into wild-type mice was stroke-protective. In conclusion, there is compelling evidence that GPIbalpha/VWF interactions and downstream signaling via phospholipase D1 (PLD1) provide new therapeutic targets in ischemic stroke.

• Platelet hyperreactivity and a prothrombotic phenotype in mice with a gain-of-function mutation in phospholipase Cgamma2.

  Authors: M Elvers, R Pozgaj, I Pleines, F May, M J E Kuijpers, J M W Heemskerk, P Yu, B Nieswandt

  Journal of thrombosis and haemostasis : JTH. 03/2010; 8(6):1353-63.

  Agonist-induced platelet activation involves different signaling pathways leading to the activation of phospholipase C (PLC) beta or PLCgamma2. Activated PLC produces inositol 1,4,5-trisphosphate andAgonist-induced platelet activation involves different signaling pathways leading to the activation of phospholipase C (PLC) beta or PLCgamma2. Activated PLC produces inositol 1,4,5-trisphosphate and diacylglycerol, which trigger Ca(2+) mobilization and the activation of protein kinase C, respectively. PLCbeta is activated downstream of Gq-coupled receptors for soluble agonists with only short interaction times in flowing blood. In contrast, PLCgamma2 becomes activated downstream of receptors that interact with immobilized ligands such as the collagen receptor glycoprotein (GP) VI or activated integrins. We speculated that PLCgamma2 activity might be optimized for sustained but submaximal signaling to control relatively slow platelet responses. To test this hypothesis, we analyzed platelets from mice heterozygous for a gain-of-function mutation in the Plcg2 gene (Plcg2(Ali5/+)). Plcg2(Ali5/+) platelets showed enhanced Ca(2+) mobilization, integrin activation, granule secretion and phosphatidylserine exposure upon GPVI or C-type lectin-like receptor-2 stimulation. Furthermore, integrin alpha(IIb)beta(3) outside-in signaling was markedly enhanced in the mutant platelets, as shown by accelerated spreading on different matrices and faster clot retraction. These defects translated into virtually unlimited thrombus formation on collagen under flow in vitro and a prothrombotic phenotype in vivo. These results demonstrate that the enzymatic activity of PLCgamma2 is tightly regulated to ensure efficient but limited platelet activation at sites of vascular injury.

• Enhanced cortical reperfusion protects coagulation factor XII-deficient mice from ischemic stroke as revealed by high-field MRI.

  Authors: M Pham, C Kleinschnitz, X Helluy, A J Bartsch, M Austinat, V C Behr, T Renné, B Nieswandt, G Stoll, M Bendszus

  NeuroImage. 12/2009;

  Intrinsic coagulation factor XII deficient (FXII(-/-)) mice are protected from ischemic stroke. To elucidate underlying mechanisms we investigated the early ischemic period in vivo by multimodalIntrinsic coagulation factor XII deficient (FXII(-/-)) mice are protected from ischemic stroke. To elucidate underlying mechanisms we investigated the early ischemic period in vivo by multimodal magnetic resonance imaging (MRI) at 17.6 Tesla. Cerebral ischemia was induced by either transient (60 min) or permanent occlusion of the middle cerebral artery (t/pMCAO). 10 FXII(-/-) mice underwent t- , 10 FXII(-/-) mice p- and 10 Wildtype (Wt) mice tMCAO. Cerebral blood flow (CBF), diffusion-weighted-imaging (DWI) and T2-relaxometry were measured at 2 h and 24 h after MCAO. Outcome measures were evaluated after motion correction and normalization to atlas space. 2 h after tMCAO CBF reduction was similar in FXII(-/-) and Wt mice extending over cortical (CBF (ml/100 g/min) 33.6+/-6.9 vs. 35.3+/-4.6, p=0.42) and subcortical regions (25.7+/-4.5 vs. 31.6+/-4.0, p=0.17). At 24 h, recovery of cortical CBF by +36% was observed only in tMCAO FXII(-/-) mice contrasting a further decrease of -48% in Wt mice after tMCAO (p=0.02, F((1,18))=6.24). In FXII(-/-) mice in which patency of the MCA was not restored (pMCAO) a further decrease of -75% was observed. Cortical reperfusion in tMCAO FXII(-/-) mice was related to a lower risk of infarction of 59% vs. 93% in Wt mice (p=0.04). Subcortical CBF was similarly decreased in both tMCAO groups (Wt and FXII(-/-)) relating to a similar risk of infarction of 89% (Wt) vs. 99% (FXII(-/-), p=0.17). Deficiency of FXII allows neocortical reperfusion after tMCAO and rescues brain tissue by this mechanism. This study supports the concept of FXII as a promising new target for stroke prevention and therapy.

• Integrins in platelet activation.

  Authors: B Nieswandt, D Varga-Szabo, M Elvers

  Journal of thrombosis and haemostasis : JTH. 08/2009; 7 Suppl 1:206-9.

  Heterodimeric receptors of the beta1 and beta3 integrin families mediate platelet adhesion and aggregation in hemostasis and thrombosis. In resting platelets, integrins are expressed in aHeterodimeric receptors of the beta1 and beta3 integrin families mediate platelet adhesion and aggregation in hemostasis and thrombosis. In resting platelets, integrins are expressed in a low-affinity state but they shift to a high-affinity state and efficiently bind their ligands in response to cellular activation. This review summarizes recent advances in understanding the functional regulation and (patho-) physiological significance of individual platelet integrins with a special focus on studies in genetically modified mice. It is now recognized that beta1 and beta3 integrins have partially redundant roles in the adhesion process and that their activation is regulated by similar mechanisms, involving Ca2+-dependent and -independent signaling events and essential functions of talin-1 and kindlin-3 in the terminal activation step.

• Calcium signaling in platelets.

  Authors: D Varga-Szabo, A. Braun, B Nieswandt

  Journal of thrombosis and haemostasis : JTH. 05/2009;

  Summary Agonist-induced elevation in cytosolic Ca(2+) concentrations is essential for platelet activation in hemostasis and thrombosis. It occurs through Ca(2+) release from intracellular stores andSummary Agonist-induced elevation in cytosolic Ca(2+) concentrations is essential for platelet activation in hemostasis and thrombosis. It occurs through Ca(2+) release from intracellular stores and Ca(2+) entry through the plasma membrane (PM). Ca(2+) store release is a well established process involving phospholipase (PL)C-mediated production of inositol-1,4,5-trisphosphate (IP(3)) which in turn releases Ca(2+) from the intracellular stores through IP(3) receptor channels. In contrast, the mechanisms controlling Ca(2+) entry and the significance of this process for platelet activation have been elucidated only very recently. In platelets, like in other non-excitable cells, the major way of Ca(2+)-entry involves the agonist induced release of cytosolic sequestered Ca(2+) followed by Ca(2+) influx through the PM, a process referred to as store-operated calcium entry (SOCE). It is now clear that stromal interaction molecule 1 (STIM1), a Ca(2+) sensor molecule in intracellular stores, and the four transmembrane channel protein Orai1 are the key players in platelet SOCE. The other major Ca(2+) entry mechanism is mediated by the direct receptor-operated calcium (ROC) channel, P2X(1). Besides these, canonical transient receptor potential channel (TRPC) 6 mediates Ca(2+) entry through the PM. This review summarizes the current knowledge of platelet Ca(2+) homeostasis with a focus on the newly identified Ca(2+) entry mechanisms.

• Complementary roles of platelets and coagulation in thrombus formation on plaques acutely ruptured by targeted ultrasound treatment: a novel intravital model.

  Authors: M J E Kuijpers, K Gilio, S Reitsma, R Nergiz-Unal, L Prinzen, S Heeneman, E Lutgens, M.A.M.J. van Zandvoort, B Nieswandt, M G A Oude Egbrink, J W M Heemskerk

  Journal of thrombosis and haemostasis : JTH. 11/2008;

  Summary Background: Atherothrombosis is a major cause of cardiovascular events. However, animal models to study this process are scarce. Objectives: We describe a first murine model of acute thrombusSummary Background: Atherothrombosis is a major cause of cardiovascular events. However, animal models to study this process are scarce. Objectives: We describe a first murine model of acute thrombus formation upon plaque rupture to study atherothrombosis by intravital fluorescence microscopy. Methods: Localized rupture of an atherosclerotic plaque in a carotid artery from Apoe(-/-) mice was induced in vivo using ultrasound. Rupture of the plaque and formation of localized thrombi was verified by two-photon laser scanning microscopy (TPLSM) in isolated arteries, and by immunohistochemistry. The thrombotic reaction was quantified by intravital fluorescence microscopy. Results: Inspection of the ultrasound-treated plaques by histochemistry and TPLSM demonstrated local damage, collagen exposure, luminal thrombus formation as well as intra-plaque intrusion of erythrocytes and fibrin. Ultrasound treatment of healthy carotid arteries resulted in endothelial damage and limited platelet adhesion. Real-time intravital fluorescence microscopy demonstrated rapid platelet deposition on plaques and formation of a single thrombus that remained subocclusive. The thrombotic process was antagonized by thrombin inhibition, or by blocking of collagen or ADP receptor pathways. Multiple thrombi were formed in 70% of mice lacking CD40L. Conclusions: Targeted rupture of murine plaques results in collagen exposure and non-occlusive thrombus formation. The thrombotic process relies on platelet activation as well as on thrombin generation and coagulation, and is sensitive to established and novel antithrombotic medication. This model provides new possibilities to study atherothrombosis in vivo.

• Platelets in atherothrombosis: lessons from mouse models.

  Authors: B Nieswandt, B Aktas, A Moers, U J H Sachs

  Journal of thrombosis and haemostasis : JTH. 09/2005; 3(8):1725-36.

  Platelets play a central role in hemostasis and thrombosis but also in the initiation of atherosclerosis, making platelet receptors and their intracellular signaling pathways important molecularPlatelets play a central role in hemostasis and thrombosis but also in the initiation of atherosclerosis, making platelet receptors and their intracellular signaling pathways important molecular targets for antithrombotic and anti-inflammatory therapy. Historically, much of the knowledge about hemostasis and thrombosis has been derived from patients suffering from bleeding and thrombotic disorders and the identification of the underlying molecular defects. In recent years, the availability of genetically modified mouse strains with defined defects in platelet function and the development of in vivo models to assess platelet-related physiologic and pathophysiologic processes have opened new ways to identify the individual roles and the interplay of platelet proteins in adhesion, activation, aggregation, secretion, and procoagulant activity in vitro and in vivo. This review will summarize key findings made by these approaches and discuss them in the context of human disease.

• Pharmacology of platelet adhesion and aggregation.

  Authors: B Nieswandt, S Offermanns

  Handbook of experimental pharmacology. 01/2004;

  At the injured vessel wall, blood platelets become activated and adhere to the subendothelial surface as well as to each other. These cellular adhesion processes are required for primary hemostasis,At the injured vessel wall, blood platelets become activated and adhere to the subendothelial surface as well as to each other. These cellular adhesion processes are required for primary hemostasis, but can also lead to thrombosis. Considerable progress has been made during recent years in understanding the molecular mechanisms underlying platelet activation and adhesion. This knowledge will drive future efforts towards the development of new antiplatelet drugs for the prevention and treatment of cardiovascular diseases.

• A PLC gamma 2-independent platelet collagen aggregation requiring functional association of GPVI and integrin alpha2beta1.

  Authors: P Mangin, C Nonne, A Eckly, P Ohlmann, M Freund, B Nieswandt, J P Cazenave, C Gachet, F Lanza

  FEBS letters. 06/2003; 542(1-3):53-9.

  The role of the phospholipase C (PLC)gamma 2 isotype in platelet activation was evaluated by studying PLC gamma 2 -/- mice. These mice have a prolonged bleeding time but their platelets respondThe role of the phospholipase C (PLC)gamma 2 isotype in platelet activation was evaluated by studying PLC gamma 2 -/- mice. These mice have a prolonged bleeding time but their platelets respond normally to non-collagenous agonists. PLC gamma 2-null platelets show residual aggregation response to collagen fibres (6% versus 74% for wild-type) with minimal granule secretion and no shape change. A delayed shape change is observed at later aggregation times. Specific activation by glycoprotein (GP)VI agonists (convulxin, collagen-related peptide and GPVI crosslinking) is, however, abolished. Antibodies against integrin alpha(2)beta(1) and GPVI each inhibit the residual collagen response, implying a role of alpha(2)beta(1) in platelet activation and a functional association with GPVI. These responses are also prevented by blocking integrin alpha(IIb)beta(3) and phosphoinositide 3-kinase, whereas aspirin treatment and ADP receptor blockade only inhibit shape change. These results provide evidence for a PLC gamma 2-independent collagen activation pathway requiring cooperation between GPVI and alpha(2)beta(1) leading to alpha(IIb)beta(3)-dependent aggregation and shape change by released ADP and thromboxane A(2).

• Differential regulation of Rho and Rac through heterotrimeric G-proteins and cyclic nucleotides.

  Authors: M P Gratacap, B Payrastre, B Nieswandt, S Offermanns

  The Journal of biological chemistry. 01/2002; 276(51):47906-13.

  Platelets were used to study the activation of Rho and Rac through G-protein-coupled receptors and its regulation by cyclic nucleotides. The thromboxane A(2) (TXA(2)) mimetic rapidly activated bothPlatelets were used to study the activation of Rho and Rac through G-protein-coupled receptors and its regulation by cyclic nucleotides. The thromboxane A(2) (TXA(2)) mimetic rapidly activated both small GTPases independently of integrin alpha(IIb)beta(3) activation., which leads to the activation of G(12)/G(13) and G(q) did not induce Rac activation in G alpha(q)-deficient platelets but was able to activate Rho, to stimulate actin polymerization and phosphatidylinositol 4,5-bisphosphate formation, and to induce shape change. Rac activation by in wild-type platelets could be blocked by chelation of intracellular Ca(2+) and was partially sensitive to apyrase and AR-C69931MX, an antagonist of the G(i)-coupled ADP receptor. Cyclic AMP, which completely blocks platelet function, inhibited the -induced activation of G(q) and G(12)/G(13) as well as of Rac and Rho. In contrast, cGMP, which has no effect on platelet shape change blocked only activation of G(q) and Rac. These data demonstrate that Rho and Rac are differentially regulated through heterotrimeric G-proteins. The G(12)/G(13)-mediated Rho activation is involved in the shape change response, whereas Rac is activated through G(q) and is not required for shape change. Cyclic AMP and cGMP differentially interfere with -induced Rho and Rac activation at least in part by selective effects on the regulation of individual G-proteins through the TXA(2) receptor.

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

  Authors: G Kauffenstein, W Bergmeier, A Eckly, P Ohlmann, C Léon, J P Cazenave, B Nieswandt, C Gachet

  FEBS letters. 10/2001; 505(2):281-90.

  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)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.

• Platelet glycoprotein V binds to collagen and participates in platelet adhesion and aggregation.

  Authors: S Moog, P Mangin, N Lenain, C Strassel, C Ravanat, S Schuhler, M Freund, M Santer, M Kahn, B Nieswandt, C Gachet, J P Cazenave, F Lanza

  Blood. 09/2001; 98(4):1038-46.

  Glycoprotein V (GPV) is a subunit of the platelet GPIb-V-IX receptor for von Willebrand factor and thrombin. GPV is cleaved from the platelet surface during activation by thrombin, but its role inGlycoprotein V (GPV) is a subunit of the platelet GPIb-V-IX receptor for von Willebrand factor and thrombin. GPV is cleaved from the platelet surface during activation by thrombin, but its role in hemostasis is still unknown. It is reported that GPV knockout mice had a decreased tendency to form arterial occluding thrombi in an intravital thrombosis model and abnormal platelet interaction with the subendothelium. In vitro, GPV-deficient platelets exhibited defective adhesion to a collagen type I-coated surface under flow or static conditions. Aggregation studies demonstrated a decreased response of the GPV-deficient platelets to collagen, reflected by an increased lag phase and reduced amplitude of aggregation. Responses to adenosine diphosphate, arachidonic acid, and the thromboxane analog U46619 were normal but were enhanced to low thrombin concentrations. The defect of GPV null platelets made them more sensitive to inhibition by the anti-GPVI monoclonal antibody (mAb) JAQ1, and this was also the case in aspirin- or apyrase-treated platelets. Moreover, an mAb (V.3) against the extracellular domain of human GPV selectively inhibited collagen-induced aggregation in human or rat platelets. V.3 injected in rats as a bolus decreased the ex vivo collagen aggregation response without affecting the platelet count. Finally, surface plasmon resonance studies demonstrated binding of recombinant soluble GPV on a collagen-coupled matrix. In conclusion, GPV binds to collagen and appears to be required for normal platelet responses to this agonist. (Blood. 2001;98:1038-1046)

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

  Authors: W Bergmeier, D Bouvard, J A Eble, R Mokhtari-Nejad, V Schulte, H Zirngibl, C Brakebusch, R Fässler, B Nieswandt

  The Journal of biological chemistry. 08/2001; 276(27):25121-6.

  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 hasAlthough 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.

• A novel viper venom metalloproteinase, alborhagin, is an agonist at the platelet collagen receptor GPVI.

  Authors: R K Andrews, E E Gardiner, N Asazuma, O Berlanga, D Tulasne, B Nieswandt, A I Smith, M C Berndt, S P Watson

  The Journal of biological chemistry. 07/2001; 276(30):28092-7.

  The interaction of platelet membrane glycoprotein VI (GPVI) with collagen can initiate (patho)physiological thrombus formation. The viper venom C-type lectin family proteins convulxin andThe interaction of platelet membrane glycoprotein VI (GPVI) with collagen can initiate (patho)physiological thrombus formation. The viper venom C-type lectin family proteins convulxin and alboaggregin-A activate platelets by interacting with GPVI. In this study, we isolated from white-lipped tree viper (Trimeresurus albolabris) venom, alborhagin, which is functionally related to convulxin because it activates platelets but is structurally different and related to venom metalloproteinases. Alborhagin-induced platelet aggregation (EC50, <7.5 microg/ml) was inhibitable by an anti-alphaIIbbeta3 antibody, CRC64, and the Src family kinase inhibitor PP1, suggesting that alborhagin activates platelets, leading to alphaIIbbeta3-dependent aggregation. Additional evidence suggested that, like convulxin, alborhagin activated platelets by a mechanism involving GPVI. First, alborhagin- and convulxin-treated platelets showed a similar tyrosine phosphorylation pattern, including a similar level of phospholipase Cgamma2 phosphorylation. Second, alborhagin induced GPVI-dependent responses in GPVI-transfected K562 and Jurkat cells. Third, alborhagin-dependent aggregation of mouse platelets was inhibited by the anti-GPVI monoclonal antibody JAQ1. Alborhagin had minimal effect on convulxin binding to GPVI-expressing cells, indicating that these venom proteins may recognize distinct binding sites. Characterization of alborhagin as a GPVI agonist that is structurally distinct from convulxin demonstrates the versatility of snake venom toxins and provides a novel probe for GPVI-dependent platelet activation.