Glycoprotein Ib-IX-V

Glycoprotein (GP) Ib-IX-V is a remarkable platelet adhesion receptor of the leucine-rich repeat family. It has evolved to fulfil its major function of initiating platelet aggregation (thrombus formation) at high-shear stress in flowing blood. In addition to binding von Willebrand factor (vWF) in subendothelial matrix or plasma to trigger platelet aggregation, GPIb-IX-V also binds counter-receptors, alphaMbeta2 (Mac-1) on neutrophils or P-selectin on activated platelets or endothelial cells. GPIb-IX-V ligands also include alpha-thrombin, clotting factors XI/XIIa, and high-molecular-weight kininogen. Interactions involving GPIb-IX-V are therefore central to vascular processes of thrombosis and inflammation, and the receptor is under intense scrutiny as a potential therapeutic target.     

Platelet - vessel wall interaction: role of blood clotting

Vascular damage initiates not only the adhesion and aggregation of blood platelets but also coagulation, which is of mixed (intrinsic and extrinsic) origin. Evidence is presented that thrombin, generated as a result of the injury, is a prerequisite for platelet aggregation. Platelets, after activation, in their turn promote coagulation. Prostaglandin I2 (PGI2 or prostacyclin) inhibits coagulation induced by damaged vascular tissue. This effect of PGI2 is mediated by the inhibition of platelets in their participation in the generation of factor Xa and thrombin. Dietary cod liver oil, by changing plasma coagulability, decreases the procoagulation activity of vessel walls, and arterial thrombosis. Another fish oil with similar effects on plasma coagulability and some other haemostatic parameters does not modify vessel wall-induced clotting, nor does it significantly lower arterial thrombosis tendency; this indicates the physiological relevance of vessel wall-induced clotting in arterial thrombus formation. Some evidence is also given for the importance of vessel wall-induced clotting in primary haemostasis.     

Tortuosity triggers platelet activation and thrombus formation in microvessels

Tortuous blood vessels are often seen in humans in association with thrombosis, atherosclerosis, hypertension, and aging. Vessel tortuosity can cause high fluid shear stress, likely promoting thrombosis. However, the underlying physical mechanisms and microscale processes are poorly understood. Accordingly, the objectives of this study were to develop and use a new computational approach to determine the effects of venule tortuosity and fluid velocity on thrombus initiation. The transport, collision, shear-induced activation, and receptor-ligand adhesion of individual platelets in thrombus formation were simulated using discrete element method. The shear-induced activation model assumed that a platelet became activated if it experienced a shear stress above a relative critical shear stress or if it contacted an activated platelet. Venules of various levels of tortuosity were simulated for a mean flow velocity of 0.10?cm s(-1), and a tortuous arteriole was simulated for a mean velocity of 0.47?cm s(-1). Our results showed that thrombus was initiated at inner walls in curved regions due to platelet activation in agreement with experimental studies. Increased venule tortuosity modified fluid flow to hasten thrombus initiation. Compared to the same sized venule, flow in the arteriole generated a higher amount of mural thrombi and platelet activation rate. The results suggest that the extent of tortuosity is an important factor in thrombus initiation in microvessels.     

Thrombokinase of the Blood as Trypsin-Like Enzyme

Thrombokinase of the blood, while resembling enterokinase in its role of activator, is more closely analogous to trypsin in its intrinsic origin. It probably arises from a plasma precursor; but it is different from plasmin (fibrinolysin). Like trypsin, thrombokinase can activate prothrombin without the aid of other factors; however, it is potentiated by platelets plus calcium. Unlike certain tissue "thromboplastins," it does not sediment appreciably in 2 hours at 85,000 g. Like trypsin, it hydrolyzes p-toluenesulfonylarginine methyl ester (TAMe). Chromatography on DEAE-cellulose separated thrombin from thrombokinase. The TAMe esterase associated with the thrombokinase fractions was largely suppressed by soybean trypsin inhibitor, while that associated with the thrombin fractions was not. Highly purified thrombokinase was used as starting material; and thrombokinase was eluted in the last major protein band. Under these conditions stepwise elution was as effective as gradient in leading to further purification. The product of 199 liters of bovine plasma was chromatographed in 1 day; and the specific activity was comparable to that attained previously by repeated electrophoretic fractionations. The assembled data suggest that the thrombokinase protein may be approaching homogeneity.     

The effect of shear stress on protein conformation: Physical forces operating on biochemical systems: The case of von Willebrand factor

Macromolecules and cells exposed to blood flow in the circulatory tree experience hydrodynamic forces that affect their structure and function. After introducing the general theory of the effects of shear forces on protein conformation, selected examples are presented in this review for biological macromolecules sensitive to shear stress. In particular, the biochemical effects of shear stress in controlling the von Willebrand Factor (VWF) conformation are extensively described. This protein, together with blood platelets, is the main actor of the early steps of primary haemostasis. Under the effect of shear forces > 30 dyn/cm², VWF unfolding occurs and the protein exhibits an extended chain conformation oriented in the general direction of the shear stress field. The stretched VWF conformation favors also a process of self aggregation, responsible for the formation of a spider web network, particularly efficient in the trapping process of flowing platelets. Thus, the effect of shear stress on conformational changes in VWF shows a close structure-function relationship in VWF for platelet adhesion and thrombus formation in arterial circulation, where high shear stress is present. The investigation of biophysical effects of shear forces on VWF conformation contributes to unraveling the molecular interaction mechanisms involved in arterial thrombosis.     

An integrated fluid–structure interaction and thrombosis model for type B aortic dissection

False lumen thrombosis (FLT) in type B aortic dissection has been associated with the progression of dissection and treatment outcome. Existing computational models mostly assume rigid wall behavior which ignores the effect of flap motion on flow and thrombus formation within the FL. In this study, we have combined a fully coupled fluid–structure interaction (FSI) approach with a shear-driven thrombosis model described by a series of convection–diffusion reaction equations. The integrated FSI-thrombosis model has been applied to an idealized dissection geometry to investigate the interaction between vessel wall motion and growing thrombus. Our simulation results show that wall compliance and flap motion can influence the progression of FLT. The main difference between the rigid and FSI models is the continuous development of vortices near the tears caused by drastic flap motion up to 4.45 mm. Flap-induced high shear stress and shear rates around tears help to transport activated platelets further to the neighboring region, thus speeding up thrombus formation during the accelerated phase in the FSI models. Reducing flap mobility by increasing the Young’s modulus of the flap slows down the thrombus growth. Compared to the rigid model, the predicted thrombus volume is 25% larger using the FSI-thrombosis model with a relatively mobile flap. Furthermore, our FSI-thrombosis model can capture the gradual effect of thrombus growth on the flow field, leading to flow obstruction in the FL, increased blood viscosity and reduced flap motion. This model is a step closer toward simulating realistic thrombus growth in aortic dissection, by taking into account the effect of intimal flap and vessel wall motion.

     

Imaging thrombus with radiolabelled monoclonal antibody to platelets.

Indium-111-hydroxyquinoline labelled platelets, though useful in the detection of thrombus, have not gained widespread use owing to the time and technical skill required for their preparation. A study was therefore conducted evaluating a new method of imaging thrombus with platelets radiolabelled with a 111In labelled monoclonal antibody, P256, directed to the platelet surface glycoprotein complex IIb/IIIa. When the number of receptors occupied by P256 was less than 3% of the total available on the platelet surface platelet function, as assessed by platelet aggregometry, was undisturbed. P256 was radiolabelled with 111In using diethylenetriaminepenta-acetic acid, which achieved a specific activity of 185 MBq (5 mCi)/mg. No impairment of immunoreactivity was detected at this specific activity. Platelets were labelled with radiolabelled monoclonal antibody in vitro in two patients at a receptor occupancy of 6% and in vivo--that is, by direct intravenous injection of P256--in six patients at a receptor occupancy of 1%. In vivo recovery and biodistribution kinetics suggested that after in vitro labelling platelets were minimally activated. The 111In kinetics recorded after intravenous P256 suggested rapid and efficient radiolabelling of platelets and gave no indication of platelet activation. Of the six patients who received intravenous P256, three had documented thrombus, two of whom gave positive results on P256 platelet scintigraphy. The third subject had chronic deep venous thrombosis and was scintigraphically negative. Imaging thrombus using a radiolabelled monoclonal antibody directed to platelets appears to offer great potential as a simple, non-invasive approach to the diagnosis of thrombosis.     

Application and validation of the lattice Boltzmann method for modelling flow-related clotting

The purpose of this paper is to present a simple clotting model, based on residence time and shear stress distribution, that can simulate the deposition over time of enzyme-activated milk in an in vitro system. Results for the model are compared with experiments exhibiting clot deposition in the region of a sharp-edged stenosis. The milk experiments have been shown to be a valuable analogue for the experimental representation of flow-induced blood clotting, particularly in the context of separation of hydrodynamic from biochemical factors. The facility to predict the flow-induced clotting of the blood analogue, in which the chemistry reduces to what is effectively a zeroth order reaction, gives confidence in this physics-based approach to simulation of the final part of the coagulation cascade. This type of study is a necessary precursor to the development of a complex, multi-factorial, biochemical model of the process of thrombosis. In addition to the clotting simulations, comparisons are reported between the computed flow patterns prior to clot deposition and flow visualisation studies. Excellent agreement of hydrodynamic parameters is reported for a Reynolds number of 100, and qualitative agreement is seen for the complex, disturbed flow occurring at a physiologically relevant Reynolds number of 550. The explicit, time-stepping lattice Boltzmann approach may have particular merit for the transitional flow at this higher Reynolds number.     

Angiotensin II via AT1 receptor accelerates arterial thrombosis in renovascular hypertensive rats.

Although there are some in vitro evidence that angiotensin II (Ang II) may promote thrombosis, there is still no data concerning effect of Ang II on arterial thrombus formation. In the present study we have investigated the influence of Ang II on electrically induced arterial thrombosis in a common carotid artery of renovascular hypertensive rats. Furthermore, we examined if Ang II effect is mediated via AT1 receptor. We measured some coagulation and fibrinolytic parameters at the same time. Since platelets play crucial role in the initiation of arterial thrombosis their contribution in the mode of Ang II action was also determined. Intravenous infusion of Ang II caused significant increase in arterial thrombus weight, which was reversed by losartan, selective AT1 receptor antagonist. The prothrombotic effect of Ang II was accompanied by increase in haemostatic and decrease in fibrinolytic potential of rat plasma. While number of data has clearly demonstrated that Ang II can augment human platelets aggregation, at least in rats, platelets were not involved in the mechanism of Ang II action. Our study shows that Ang II via AT1 receptor accelerates arterial thrombosis in renovascular hypertensive rat, therefore may be considered as a risk factor of myocardial infarction or stroke.     

A Novel Model of Intravital Platelet Imaging Using CD41-ZsGreen1 Transgenic Rats

Platelets play pivotal roles in both hemostasis and thrombosis. Although models of intravital platelet imaging are available for thrombosis studies in mice, few are available for rat studies. The present effort aimed to generate fluorescent platelets in rats and assess their dynamics in a rat model of arterial injury. We generated CD41-ZsGreen1 transgenic rats, in which green fluorescence protein ZsGreen1 was expressed specifically in megakaryocytes and thus platelets. The transgenic rats exhibited normal hematological and biochemical values with the exception of body weight and erythroid parameters, which were slightly lower than those of wild-type rats. Platelet aggregation, induced by 20 μM ADP and 10 μg/ml collagen, and blood clotting times were not significantly different between transgenic and wild-type rats. Saphenous arteries of transgenic rats were injured with 10% FeCl₃, and the formation of fluorescent thrombi was evaluated using confocal microscopy. FeCl₃ caused time-dependent increases in the mean fluorescence intensity of injured arteries of vehicle-treated rats. Prasugrel (3 mg/kg, p.o.), administered 2 h before FeCl₃, significantly inhibited fluorescence compared with vehicle-treated rats (4.5 ± 0.4 vs. 14.9 ± 2.4 arbitrary fluorescence units at 30 min, respectively, n = 8, P = 0.0037). These data indicate that CD41-ZsGreen1 transgenic rats represent a useful model for intravital imaging of platelet-mediated thrombus formation and the evaluation of antithrombotic agents.     

重组活性小鼠组织因子及其阻断性单抗的制备与应用

组织因子是一种位于细胞膜上的糖蛋白,是外源性凝血过程的关键启动因子,近年来其在肿瘤细胞迁移等其他过程中的重要作用也已逐渐被揭示．构建了融合有His标签的小鼠组织因子胞外区段重组蛋白基因,利用昆虫杆状病毒蛋白表达系统成功表达并得到大量可溶性重组小鼠组织因子．利用血浆凝集实验和鼠尾流血时间实验对此重组小鼠组织因子进行的活性检测表明,此重组蛋白具有良好的生物活性,可以引起血浆凝血或缩短鼠尾流血时间．同时,利用此重组蛋白为抗原,制备了小鼠组织因子的小鼠源功能阻断性单克隆抗体,在血浆凝集实验中证明其对小鼠组织因子的活性有明显抑制作用．利用此阻断性单抗,成功地在小鼠深静脉血栓模型中减轻了血栓形成,证明组织因子在深静脉血栓的病程发展中起重要作用,这也是组织因子阻断性单抗在此类动物模型中的首次成功应用．通过此项工作,成功地建立了大量制备具有良好生物活性的重组小鼠组织因子蛋白的方法,并进而得到了小鼠组织因子功能阻断性单抗,为利用各种小鼠动物模型对组织因子在各项生命活动中的作用进行深入研究奠定了良好的基础．     

Slowness of blood flow and resultant thrombus formation in cerebral aneurysms

It is not yet fully understood what causes cerebral aneurysms to rupture. Although no definitive conclusion has been reached, it is considered that there are haemodynamic, biochemical and physiological factors contributing to rupture. Numerical techniques seem promising for investigation of this multi-physical phenomenon. In fact, recent intensive numerical studies with computational fluid dynamics have revealed detailed haemodynamic features of the flow in cerebral aneurysms such as velocity, pressure and wall shear stress distributions. It is, therefore, expected that biochemical and physiological aspects of aneurysmal rupture will also be actively investigated using numerical approaches. Considering this background, the authors have been working on modelling of thrombus formation in cerebral aneurysms caused by stagnant blood flow, because many studies have suggested that slow blood flow and resulting low wall shear stress are connected with rupture. Firstly, in this review paper, slowness of the intra-aneurysmal flow is reviewed with an energy balance theory, and secondly, thrombus formation in cerebral bifurcation aneurysms is discussed from the viewpoint of numerical modelling. A computational result obtained by application of the authors’ platelet aggregation–adhesion model is also provided.     

How it all starts: Initiation of the clotting cascade

Abstract

The plasma coagulation system in mammalian blood consists of a cascade of enzyme activation events in which serine proteases activate the proteins (proenzymes and procofactors) in the next step of the cascade via limited proteolysis. The ultimate outcome is the polymerization of fibrin and the activation of platelets, leading to a blood clot. This process is protective, as it prevents excessive blood loss following injury (normal hemostasis). Unfortunately, the blood clotting system can also lead to unwanted blood clots inside blood vessels (pathologic thrombosis), which is a leading cause of disability and death in the developed world. There are two main mechanisms for triggering the blood clotting, termed the tissue factor pathway and the contact pathway. Only one of these pathways (the tissue factor pathway) functions in normal hemostasis. Both pathways, however, are thought to contribute to thrombosis. An emerging concept is that the contact pathway functions in host pathogen defenses. This review focuses on how the initiation phase of the blood clotting cascade is regulated in both pathways, with a discussion of the contributions of these pathways to hemostasis versus thrombosis.     

Effect of blood thrombokinase,as influenced by soy bean trypsin inhibitor,ultracentrifugation, and accessory factors

1. Crystallized soy bean trypsin inhibitor, at a concentration of 100 microg./ml., suppressed the production of thrombin from a mixture of prothrombin and blood thrombokinase. The experiment was performed in the presence of 0.011 M oxalate, in order to minimize the possibility of participation by accessory factors which require ionic calcium. The results are in accord with the view that thrombokinase is a trypsin-like enzyme. 2. When a solution of blood thrombokinase was centrifuged at 85,000 g for 120 minutes, almost all the activity remained in the supernate. This supernate activated the supernate from a prothrombin solution which had been similarly centrifuged. The activation of prothrombin by thrombokinase can proceed in the absence of material completely sedimentable in 120 minutes at 85,000 g. 3. An "accelerator" reagent was prepared by treating bovine serum with barium carbonate, and then passing the serum through a column of diatomaceous earth. This "accelerator" was used together with prothrombin, blood thrombokinase, Howell's cephalin, and calcium chloride to compose a five-reagent thrombin-producing system. In this system, no thrombin was produced without thrombokinase. On the other hand, thrombin was produced from prothrombin and thrombokinase, even when all the other reagents were omitted. When calcium was omitted, thrombokinase was able to function; but cephalin and the "accelerator" reagent were ineffective. 4. Quantitative tests indicated that the "accelerator" reagent exerted an effect distinct from those of thrombokinase and cephalin. However, it is not certain whether the "accelerator" reagent functioned as an accessory factor, as a potential source of more thrombokinase, or both. In the experiments reported, thrombokinase was primary to, or necessary for, the effect of "accelerator." 5. The effectiveness of thrombokinase was multiplied a hundred times or more, when complemented by calcium, cephalin, and "accelerator" reagent. Ionic calcium was a necessary component of this complementing system. This may help to explain why removal of calcium ions keeps blood fluid, even though thrombokinase, by itself, is little influenced either by calcium ions or by oxalate.     

von Willebrand factor unfolding mediates platelet deposition in a model of high-shear thrombosis

Thrombosis under high-shear conditions is mediated by the mechanosensitive blood glycoprotein von Willebrand factor (vWF). vWF unfolds in response to strong flow gradients and facilitates rapid recruitment of platelets in flowing blood. While the thrombogenic effect of vWF is well recognized, its conformational response in complex flows has largely been omitted from numerical models of thrombosis. We recently presented a continuum model for the unfolding of vWF, where we represented vWF transport and its flow-induced conformational change using convection-diffusion-reaction equations. Here, we incorporate the vWF component into our multi-constituent model of thrombosis, where the local concentration of stretched vWF amplifies the deposition rate of free-flowing platelets and reduces the shear cleaning of deposited platelets. We validate the model using three benchmarks: in vitro model of atherothrombosis, a stagnation point flow, and the PFA-100, a clinical blood test commonly used for screening for von Willebrand disease (vWD). The simulations reproduced the key aspects of vWF-mediated thrombosis observed in these experiments, such as the thrombus location, thrombus growth dynamics, and the effect of blocking platelet-vWF interactions. The PFA-100 simulations closely matched the reported occlusion times for normal blood and several hemostatic deficiencies, namely, thrombocytopenia, vWD type 1, and vWD type 3. Overall, this multi-constituent model of thrombosis enables macro-scale 3D simulations of thrombus formation in complex geometries over a wide range of shear rates and accounts for qualitative and quantitative hemostatic deficiencies in patient blood.     

Survival and detectability bias of avian fence collision surveys in sagebrush steppe

We used female ring-necked pheasant (Phasianus colchicus) carcasses as surrogates for greater sage-grouse (Centrocercus urophasianus) to study factors influencing survival and detection bias associated with avian fence collision surveys in southern Idaho, USA, during spring 2009. We randomly placed 50 pheasant carcasses on each of 2 study areas, estimated detection probability during fence-line surveys, and monitored survival and retention of carcasses and their associated sign over a 31-day period. Survival modeling suggested site and habitat features had little impact on carcass survival, and constant survival models were most supported by the data. Model averaged carcass daily survival probability was low on both study areas and ranged from 0.776 to 0.812. Survival of all carcass sign varied strongly by location, and the top sign survival model included a site effect parameter. Model averaged daily survival probability for collision sign on the 2 study sites ranged from 0.863 to 0.988 and varied between sites. Logistic regression modeling indicated detection probability of carcasses during fence-line surveys for avian collision victims was influenced by habitat type and microsite shrub height at the carcass location. Carcasses located in big sagebrush (Artemisia tridentata) habitats were detected at a lower rate (0.36) than carcasses in little (A. arbuscula) and black sagebrush (A. nova) habitats (0.71). Increasing shrub height at the carcass location from the little sagebrush mean of 16.5 cm to the big sagebrush mean of 36.0 cm reduced detection probability by approximately 30%. Avian fence collision surveys in sagebrush-steppe habitats should be conducted at ≤2-week sampling intervals to reduce the impact of survival bias on collision rate estimates. Two-week sampling intervals may be too long in areas with low carcass and sign survival, therefore survival rates should be estimated on all study areas to determine the appropriate sampling interval duration. Researchers should be aware of the effects of local vegetation on detection probabilities, and methods to correct detection probabilities based on collision site attributes should be applied to ensure more accurate collision rate estimates. Additionally, caution should be used when aggregating or comparing uncorrected collision data from areas with differing vegetation, as detection probabilities are likely different between sites. © 2011 The Wildlife Society.     

Serotonin metabolism in thrombocytes and microvascular hemostasis in spontaneous arterial hypertension

Serotonin content and accumulation in platelets and its release from them, as well as changes in thrombus formation in mesenteric arterioles and venules of the small intestine have been investigated in control rats and rats with spontaneous hypertension (SHR). Serotonin accumulation in platelets was determined upon its incubation with platelets. Disodium ADP salt was used as an inductor of release. Laser-induced thrombosis was caused by microvessels exposure to impulse laser irradiation. The control animals revealed a significant difference between the initial serotonin platelet level and serotonin level upon incubation and release; in values, the values of basic thrombus-forming parameters were higher than in arterioles. In SHR there is a decrease in biogenic amine content in platelets, a depression in its accumulation and release, an increase in the time of thrombus growth, its size up to the separation of the first embolus and its length along the vascular wall. It is concluded that spontaneous hypertension is characterized by decreased functional activity of platelets and depressed resistance of arterioles and venules to thrombus formation.     

PI 3-kinase p110beta: a new target for antithrombotic therapy

Platelet activation at sites of vascular injury is essential for the arrest of bleeding; however, excessive platelet accumulation at regions of atherosclerotic plaque rupture can result in the development of arterial thrombi, precipitating diseases such as acute myocardial infarction and ischemic stroke. Rheological disturbances (high shear stress) have an important role in promoting arterial thrombosis by enhancing the adhesive and signaling function of platelet integrin alpha(IIb)beta(3) (GPIIb-IIIa). In this study we have defined a key role for the Type Ia phosphoinositide 3-kinase (PI3K) p110beta isoform in regulating the formation and stability of integrin alpha(IIb)beta(3) adhesion bonds, necessary for shear activation of platelets. Isoform-selective PI3K p110beta inhibitors have been developed which prevent formation of stable integrin alpha(IIb)beta(3) adhesion contacts, leading to defective platelet thrombus formation. In vivo, these inhibitors eliminate occlusive thrombus formation but do not prolong bleeding time. These studies define PI3K p110beta as an important new target for antithrombotic therapy.     

Platelet adhesive dynamics. Part I: characterization of platelet hydrodynamic collisions and wall effects

Abnormally high shear stresses encountered in vivo induce spontaneous activation of blood platelets and formation of aggregates, even in the absence of vascular injury. A three-dimensional multiscale computational model—platelet adhesive dynamics—is developed and applied in Part I and Part II articles to elucidate key biophysical aspects of GPIbα-von-Willebrand-factor-mediated interplatelet binding that characterizes the onset of shear-induced platelet aggregation. In this article, the hydrodynamic effects of the oblate spheroidal shape of platelets and proximity of a plane wall on the nature of cell-cell collisions are systematically investigated. Physical quantities characterizing the adhesion probabilities between colliding platelet surfaces for the entire range of near-wall encounters between two platelets are obtained for application in platelet adhesive dynamics simulations of platelet aggregation explored in a companion article. The technique for matching simulation predictions of interplatelet binding efficiency to experimentally determined efficiencies is also described. Platelet collision behavior is found to be strikingly different from that of spheres, both close to and far from a bounding wall. Our results convey the significant effects that particle shape and presence of a bounding wall have on the particle trajectories and collision mechanisms, collision characteristics such as collision time and contact area, and collision frequency.     

Functional Divergence of Platelet Protein Kinase C (PKC) Isoforms in Thrombus Formation on Collagen

Arterial thrombosis, a major cause of myocardial infarction and stroke, is initiated by activation of blood platelets by subendothelial collagen. The protein kinase C (PKC) family centrally regulates platelet activation, and it is becoming clear that the individual PKC isoforms play distinct roles, some of which oppose each other. Here, for the first time, we address all four of the major platelet-expressed PKC isoforms, determining their comparative roles in regulating platelet adhesion to collagen and their subsequent activation under physiological flow conditions. Using mouse gene knock-out and pharmacological approaches in human platelets, we show that collagen-dependent α-granule secretion and thrombus formation are mediated by the conventional PKC isoforms, PKCα and PKCβ, whereas the novel isoform, PKCθ, negatively regulates these events. PKCδ also negatively regulates thrombus formation but not α-granule secretion. In addition, we demonstrate for the first time that individual PKC isoforms differentially regulate platelet calcium signaling and exposure of phosphatidylserine under flow. Although platelet deficient in PKCα or PKCβ showed reduced calcium signaling and phosphatidylserine exposure, these responses were enhanced in the absence of PKCθ. In summary therefore, this direct comparison between individual subtypes of PKC, by standardized methodology under flow conditions, reveals that the four major PKCs expressed in platelets play distinct non-redundant roles, where conventional PKCs promote and novel PKCs inhibit thrombus formation on collagen.