Targeting shear gradient activated von Willebrand factor by the novel single-chain antibody A1 reduces occlusive thrombus formation in vitro.

Intraluminal thrombus formation precipitates conditions such as acute myocardial infarction and disturbs local blood flow resulting in areas of rapidly changing blood flow velocities and steep gradients of blood shear rate. Shear rate gradients are known to be pro-thrombotic with an important role for the shear-sensitive plasma protein von Willebrand factor (VWF). Here, we developed a single-chain antibody (scFv) that targets a shear gradient specific conformation of VWF to specifically inhibit platelet adhesion at sites of SRGs but not in areas of constant shear. Microfluidic flow channels with stenotic segments were used to create shear rate gradients during blood perfusion. VWF-GPIbα interactions were increased at sites of shear rate gradients compared to constant shear rate of matched magnitude. The scFv-A1 specifically reduced VWF-GPIbα binding and thrombus formation at sites of SRGs but did not block platelet deposition and aggregation under constant shear rate in upstream sections of the channels. Significantly, the scFv A1 attenuated platelet aggregation only in the later stages of thrombus formation. In the absence of shear, direct binding of scFv-A1 to VWF could not be detected and scFV-A1 did not inhibit ristocetin induced platelet agglutination. We have exploited the pro-aggregatory effects of SRGs on VWF dependent platelet aggregation and developed the shear-gradient sensitive scFv-A1 antibody that inhibits platelet aggregation exclusively at sites of shear rate gradients. The lack of VWF inhibition in non-stenosed vessel segments places scFV-A1 in an entirely new class of anti-platelet therapy for selective blockade of pathological thrombus formation while maintaining normal haemostasis.

Targeting Nanotechnologies for the Treatment of Thrombosis and Cardiovascular Disease.

Thrombosis is characterized by the formation of in vivo blood clots that are localized within arterial or venous blood vessels. These thrombi form beyond the need for physiologically healthy hemostatic responses and can lead to significant medical issues for affected individuals. Unfortunately, the existing standard-of-care therapies for treating thrombosis are systemic in their therapeutic design; therefore, they interfere with the patient's physiological hemostasis. Examples of the severe clinical side effects commonly associated with currently available therapies include, but are not limited to, bleeding complications. Therefore, there is a profound demand for novel therapeutic interventions that can circumvent these debilitating complications, while offering improved therapeutic efficacy. Recent advancements in nanotechnology present an opportunity to develop novel and improved drug delivery systems to meet this clinical demand. Preclinical investigations have begun to uncover the potential of nanotechnology, particularly in the treatment of thrombosis and also in nonhemostatic cardiovascular diseases. This article reviews recent preclinical studies aimed at developing a diverse array of different nanotechnologies for treating thrombosis as well as heart diseases. This review will also outline the limitations with current nanotechnologies and what challenges need to be overcome to translate these novel therapies to the clinic.

Shear-Dependent Platelet Aggregation: Mechanisms and Therapeutic Opportunities.

Cardiovascular diseases (CVD) are the number one cause of morbidity and death worldwide. As estimated by the WHO, the global death rate from CVD is 31% wherein, a staggering 85% results from stroke and myocardial infarction. Platelets, one of the key components of thrombi, have been well-investigated over decades for their pivotal role in thrombus development in healthy as well as diseased blood vessels. In hemostasis, when a vascular injury occurs, circulating platelets are arrested at the site of damage, where they are activated and aggregate to form hemostatic thrombi, thus preventing further bleeding. However, in thrombosis, pathological activation of platelets occurs, leading to uncontrolled growth of a thrombus, which in turn can occlude the blood vessel or embolize, causing downstream ischemic events. The molecular processes causing pathological thrombus development are in large similar to the processes controlling physiological thrombus formation. The biggest challenge of anti-thrombotics and anti-platelet therapeutics has been to decouple the pathological platelet response from the physiological one. Currently, marketed anti-platelet drugs are associated with major bleeding complications for this exact reason; they are not effective in targeting pathological thrombi without interfering with normal hemostasis. Recent studies have emphasized the importance of shear forces generated from blood flow, that primarily drive platelet activation and aggregation in thrombosis. Local shear stresses in obstructed blood vessels can be higher by up to two orders of magnitude as compared to healthy vessels. Leveraging abnormal shear forces in the thrombus microenvironment may allow to differentiate between thrombosis and hemostasis and develop shear-selective anti-platelet therapies. In this review, we discuss the influence of shear forces on thrombosis and the underlying mechanisms of shear-induced platelet activation. Later, we summarize the therapeutic approaches to target shear-sensitive platelet activation and pathological thrombus growth, with a particular focus on the shear-sensitive protein von Willebrand Factor (VWF). Inhibition of shear-specific platelet aggregation and targeted drug delivery may prove to be much safer and efficacious approaches over current state-of-the-art antithrombotic drugs in the treatment of cardiovascular diseases.

Thrombosis in diabetes: a shear flow effect?

Cardiovascular events are the major cause of morbidity and mortality in Type 2 diabetes (T2D). This condition is associated with heightened platelet reactivity, contributing to increased atherothrombotic risk. Indeed, individuals with diabetes respond inadequately to standard antiplatelet therapy. Furthermore, they often experience recurrent events as well as side effects that include excess bleeding. This highlights the need for identification of novel regulators of diabetes-associated thrombosis to target for therapeutic intervention. It is well established that platelet aggregation, a process essential for thrombus formation, is tightly regulated by shear stress; however, the mechanisms underlying shear activation of platelets, particularly in the setting of diabetes, are still poorly understood. This review will address the limitations of current diagnostic systems to assess the importance of shear stress in the regulation of thrombus formation in T2D, and the inability to recapitulate the pro-thrombotic phenotype seen clinically in the setting of T2D. Moreover, we will discuss recent findings utilizing new technologies to define the importance of shear stress in thrombus formation and their potential application to the setting of diabetes. Finally, we will discuss the potential of targeting shear-dependent mechanisms of thrombus formation as a novel therapeutic approach in the setting of T2D.

Neutrophil-derived S100 calcium-binding proteins A8/A9 promote reticulated thrombocytosis and atherogenesis in diabetes.

Platelets play a critical role in atherogenesis and thrombosis-mediated myocardial ischemia, processes that are accelerated in diabetes. Whether hyperglycemia promotes platelet production and whether enhanced platelet production contributes to enhanced atherothrombosis remains unknown. Here we found that in response to hyperglycemia, neutrophil-derived S100 calcium-binding proteins A8/A9 (S100A8/A9) interact with the receptor for advanced glycation end products (RAGE) on hepatic Kupffer cells, resulting in increased production of IL-6, a pleiotropic cytokine that is implicated in inflammatory thrombocytosis. IL-6 acts on hepatocytes to enhance the production of thrombopoietin, which in turn interacts with its cognate receptor c-MPL on megakaryocytes and bone marrow progenitor cells to promote their expansion and proliferation, resulting in reticulated thrombocytosis. Lowering blood glucose using a sodium-glucose cotransporter 2 inhibitor (dapagliflozin), depleting neutrophils or Kupffer cells, or inhibiting S100A8/A9 binding to RAGE (using paquinimod), all reduced diabetes-induced thrombocytosis. Inhibiting S100A8/A9 also decreased atherogenesis in diabetic mice. Finally, we found that patients with type 2 diabetes have reticulated thrombocytosis that correlates with glycated hemoglobin as well as increased plasma S100A8/A9 levels. These studies provide insights into the mechanisms that regulate platelet production and may aid in the development of strategies to improve on current antiplatelet therapies and to reduce cardiovascular disease risk in diabetes.

Multi-photon microscopy in cardiovascular research.

Multiphoton laser scanning microscopy has proven profound value for ex vivo 3D histology and in vivo imaging of motionless tissue. The development of triggering systems and fast imaging methods, combined with advanced preparation procedures solved the challenging task of intravital imaging of the fast pulsating heart and major arteries in animals and further increased the popularity of intravital multiphoton imaging in cardiovascular research. This review article will highlight the potential of multiphoton microscopy for the visualization and characterization of dynamical and structural processes involved in cardiac and vascular diseases, both in an ex vivo and an intravital animal setting. Examples will be given how multiphoton microscopy can be applied to imaging of atherosclerotic plaque development and progression at subcellular level as well as to intravital imaging of inflammatory processes in the heart. In addition to highlighting the potential of multiphoton microscopy in preclinical cardiovascular research, we will discuss how this tool and its applications may be clinically translated to support disease diagnosis and therapy in patients.

Multifunctional Thrombin-Activatable Polymer Capsules for Specific Targeting to Activated Platelets.

Smart poly(2-oxazoline) (POx)-based multifunctional polymer capsules that specifically target glycoprotein (GP) IIb/IIIa on the surface of activated platelets are degraded by the serine protease thrombin and release the urokinase plasminogen activator loaded into the polymer capsules, only in the area of acute thrombosis.

A versatile approach for the site-specific modification of recombinant antibodies using a combination of enzyme-mediated bioconjugation and click chemistry.

A unique two-step modular system for site-specific antibody modification and conjugation is reported. The first step of this approach uses enzymatic bioconjugation with the transpeptidase Sortase A for incorporation of strained cyclooctyne functional groups. The second step of this modular approach involves the azide-alkyne cycloaddition click reaction. The versatility of the two-step approach has been exemplified by the selective incorporation of fluorescent dyes and a positron-emitting copper-64 radiotracer for fluorescence and positron-emission tomography imaging of activated platelets, platelet aggregates, and thrombi, respectively. This flexible and versatile approach could be readily adapted to incorporate a large array of tailor-made functional groups using reliable click chemistry whilst preserving the activity of the antibody or other sensitive biological macromolecules.

Towards effective and safe thrombolysis and thromboprophylaxis: preclinical testing of a novel antibody-targeted recombinant plasminogen activator directed against activated platelets.

RATIONALE: Fibrinolysis is a valuable alternative for the treatment of myocardial infarction when percutaneous coronary intervention is not available in a timely fashion. For acute ischemic stroke, fibrinolysis is the only treatment option with a very narrow therapeutic window. Clinically approved thrombolytics have significant drawbacks, including bleeding complications. Thus their use is highly restricted, leaving many patients untreated. OBJECTIVE: We developed a novel targeted fibrinolytic drug that is directed against activated platelets. METHODS AND RESULTS: We fused single-chain urokinase plasminogen activator (scuPA) to a small recombinant antibody (scFvSCE5), which targets the activated form of the platelet-integrin glycoprotein IIb/IIIa. Antibody binding and scuPA activity of this recombinant fusion protein were on par with the parent molecules. Prophylactic in vivo administration of scFvSCE5-scuPA (75 U/g body weight) prevented carotid artery occlusion after ferric chloride injury in a plasminogen-dependent process compared with saline (P<0.001), and blood flow recovery was similar to high-dose nontargeted urokinase (500 U/g body weight). Tail bleeding time was significantly prolonged with this high dose of nontargeted urokinase, but not with equally effective targeted scFvSCE5-scuPA at 75 U/g body weight. Real-time in vivo molecular ultrasound imaging demonstrates significant therapeutic reduction of thrombus size after administration of 75 U/g body weight scFvSCE5-scuPA as compared with the same dose of a mutated, nontargeting scFv-scuPA or vehicle. The ability of scFvSCE5-scuPA to lyse thrombi was lost in plasminogen-deficient mice, but could be restored by intravenous injection of plasminogen. CONCLUSIONS: Targeting of scuPA to activated glycoprotein IIb/IIIa allows effective thrombolysis and the potential novel use as a fibrinolytic agent for thromboprophylaxis without bleeding complications.

Dok-2 adaptor protein regulates the shear-dependent adhesive function of platelet integrin αIIbß3 in mice.

The Dok proteins are a family of adaptor molecules that have a well defined role in regulating cellular migration, immune responses, and tumor progression. Previous studies have demonstrated that Doks-1 to 3 are expressed in platelets and that Dok-2 is tyrosine-phosphorylated downstream of integrin αIIbß3, raising the possibility that it participates in integrin αIIbß3 outside-in signaling. We demonstrate that Dok-2 in platelets is primarily phosphorylated by Lyn kinase. Moreover, deficiency of Dok-2 leads to dysregulated integrin αIIbß3-dependent cytosolic calcium flux and phosphatidylinositol(3,4)P2 accumulation. Although agonist-induced integrin αIIbß3 affinity regulation was unaltered in Dok-2(-/-) platelets, Dok-2 deficiency was associated with a shear-dependent increase in integrin αIIbß3 adhesive function, resulting in enhanced platelet-fibrinogen and platelet-platelet adhesive interactions under flow. This increase in adhesion was restricted to discoid platelets and involved the shear-dependent regulation of membrane tethers. Dok-2 deficiency was associated with an increased rate of platelet aggregate formation on thrombogenic surfaces, leading to accelerated thrombus growth in vivo. Overall, this study defines an important role for Dok-2 in regulating biomechanical adhesive function of discoid platelets. Moreover, they define a previously unrecognized prothrombotic mechanism that is not detected by conventional platelet function assays.

A multimode-TIRFM and microfluidic technique to examine platelet adhesion dynamics.

Fluorescence microscopy techniques have provided important insights into the structural and signalling events occurring during platelet adhesion under both static and blood flow conditions. However, due to limitations in sectioning ability and sensitivity these techniques are restricted in their capacity to precisely image the adhesion footprint of spreading platelets. In particular, investigation of platelet adhesion under hemodynamic shear stress requires an imaging platform with high spatial discrimination and sensitivity and rapid temporal resolution. This chapter describes in detail a multimode imaging approach combining total internal reflection fluorescence microscopy (TIRFM) with high speed epifluorescence and differential interference contrast (DIC) microscopy along with a novel microfluidic perfusion system developed in our laboratory to examine platelet membrane adhesion dynamics under static and flow conditions.

The CXCR1/2 ligand NAP-2 promotes directed intravascular leukocyte migration through platelet thrombi.

Thrombosis promotes leukocyte infiltration into inflamed tissues, leading to organ injury in a broad range of diseases; however, the mechanisms by which thrombi guide leukocytes to sites of vascular injury remain ill-defined. Using mouse models of endothelial injury (traumatic or ischemia reperfusion), we demonstrate a distinct process of leukocyte recruitment, termed "directed intravascular migration," specifically mediated by platelet thrombi. Single adherent platelets and platelet aggregates stimulated leukocyte shape change at sites of endothelial injury; however, only thrombi were capable of inducing directed intravascular leukocyte migration. Leukocyte recruitment and migration induced by platelet thrombi occurred most prominently in veins but could also occur in arteries following ischemia-reperfusion injury. In vitro studies demonstrated a major role for platelet-derived NAP-2 (CXCL-7) and its CXCR1/2 receptor in regulating leukocyte polarization and motility. In vivo studies demonstrated the presence of an NAP-2 chemotactic gradient within the thrombus body. Pharmacologic blockade of CXCR1/2 as well as genetic deletion of NAP-2 markedly reduced leukocyte shape change and intrathrombus migration. These studies define a distinct process of leukocyte migration that is initiated by homotypic adhesive interactions between platelets, leading to the development of an NAP-2 chemotactic gradient within the thrombus body that guides leukocytes to sites of vascular injury.

Atherosclerotic geometries exacerbate pathological thrombus formation poststenosis in a von Willebrand factor-dependent manner.

Rupture of a vulnerable atherosclerotic plaque causes thrombus formation and precipitates cardiovascular diseases. In addition to the thrombogenic content of a plaque, also the hemodynamic microenvironment plays a major role in thrombus formation. How the altered hemodynamics around a plaque promote pathological thrombus formation is not well understood. In this study, we provide evidence that plaque geometries result in fluid mechanical conditions that promote platelet aggregation and thrombus formation by increased accumulation and activity of von Willebrand factor (vWF) at poststenotic sites. Resonant-scanning multiphoton microscopy revealed that in vivo arterial stenosis of a damaged carotid artery markedly increased platelet aggregate formation in the stenotic outlet region. Complementary in vitro studies using microfluidic stenotic chambers, designed to mimic the flow conditions in a stenotic artery, showed enhanced platelet aggregation in the stenotic outlet region at 60-80% channel occlusion over a range of input wall shear rates. The poststenotic thrombus formation was critically dependent on bloodborne vWF and autocrine platelet stimulation. In stenotic chambers containing endothelial cells, flow provoked increased endothelial vWF secretion in the stenotic outlet region, contributing to exacerbated platelet aggregation. Taken together, this study identifies a role for the shear-sensitive protein vWF in transducing hemodynamic forces that are present around a stenosis to a prothrombogenic microenvironment resulting in spatially confined and exacerbated platelet aggregation in the stenosis outlet region. The developed stenotic microfluidic chamber offers a realistic platform for in vitro evaluation of shear-dependent thrombus formation in the setting of atherosclerosis.

Monitoring in vitro thrombus formation with novel microfluidic devices.

Cardiovascular disease is a major cause of mortality globally and is subject to ongoing research to improve clinical treatment. It is established that activation of platelets and coagulation are central to thrombosis, yet at different extents in the arterial and venous system. In vitro perfusion chamber technology has contributed significant knowledge on the function of platelets in the thrombotic process under shear conditions. Recent efforts to downscale this technique with a variety of microfluidic devices has opened new possibilities to study this process under precisely controlled flow conditions. Such microfluidic devices possess the capability to execute platelet function tests more quickly than current assays, while using small blood samples. Gradually becoming available to the clinic now, they may provide a new means to manage the treatment of cardiovascular diseases, although accurate validation studies still are missing. This review highlights the progress that has been made in monitoring aspects of thrombus formation using microfluidic devices.

Evidence of platelet activation at medically used hypothermia and mechanistic data indicating ADP as a key mediator and therapeutic target.

OBJECTIVE: Hypothermia is used in various clinical settings to inhibit ischemia-related organ damage. However, prothrombotic effects have been described as potential side effects. This study aimed to elucidate the mechanism of hypothermia-induced platelet activation and subsequent prothrombotic events and to develop preventative pharmacological strategies applicable during clinically used hypothermia. METHODS AND RESULTS: Platelet function was investigated ex vivo and in vivo at clinically used hypothermia (28°C/18°C). Hypothermic mice demonstrated increased expression of platelet activation marker P-selectin, platelet-leukocyte aggregate formation, and thrombocytopenia. Intravital microscopy of FeCl(3)-injured murine mesenteric arteries revealed increased platelet thrombus formation with hypothermia. Ex vivo flow chamber experiments indicated increased platelet-fibrinogen adhesion under hypothermia. We show that hypothermia results in reduced ADP hydrolysis via reduction of CD39 (E-NTPDase1) activity, resulting in increased levels of ADP and subsequent augmented primary and secondary platelet activation. In vivo administration of ADP receptor P(2)Y(12) antagonists and recombinant soluble CD39 prevented hypothermia-induced thrombus formation and thrombocytopenia, respectively. CONCLUSIONS: The platelet agonist ADP plays a key role in hypothermia-induced platelet activation. Inhibition of receptor binding or hydrolysis of ADP has the potential to protect platelets against hypothermia-induced activation. Our findings provide a rational basis for further evaluation of novel antithrombotic strategies in clinically applied hypothermia.

Structural and hydrodynamic simulation of an acute stenosis-dependent thrombosis model in mice.

Platelet activation under blood flow is thought to be critically dependent on the autologous secretion of soluble platelet agonists (chemical activators) such as ADP and thromboxane. However, recent evidence challenging this model suggests that platelet activation can occur independent of soluble agonist signalling, in response to the mechanical effects of micro-scale shear gradients. A key experimental tool utilized to define the effect of shear gradients on platelet aggregation is the murine intravital microscopy model of platelet thrombosis under conditions of acute controlled arteriolar stenosis. This paper presents a computational structural and hydrodynamic simulation of acute stenotic blood flow in the small bowel mesenteric vessels of mice. Using a homogeneous fluid at low Reynolds number (0.45) we investigated the relationship between the local hydrodynamic strain-rates and the severity of arteriolar stensosis. We conclude that the critical rates of blood flow acceleration and deceleration at sites of artificially induced stenosis (vessel side-wall compression or ligation) are a function of tissue elasticity. By implementing a structural simulation of arteriolar side wall compression, we present a mechanistic model that provides accurate simulations of stenosis in vivo and allows for predictions of the effects on local haemodynamics in the murine small bowel mesenteric thrombosis model.

Platelet recruitment to the inflamed glomerulus occurs via an alphaIIbbeta3/GPVI-dependent pathway.

Recruitment of leukocytes to glomeruli is fundamental to the pathogenesis of many forms of glomerulonephritis. In a model of glomerulonephritis induced by in situ immune complex deposition, we previously observed that, in addition to leukocytes, platelets accumulate in glomerular capillaries, where they contribute to leukocyte recruitment. However, the mechanisms of platelet recruitment and the role of platelet-expressed P-selectin in leukocyte recruitment require further investigation. We used intravital microscopy to examine the mechanisms of platelet and leukocyte recruitment to glomeruli of mice following administration of an antibody against the glomerular basement membrane (anti-GBM antibody). Platelet recruitment was initiated within five minutes of administration of anti-GBM antibody. This was unaltered by inhibition of platelet GPIbalpha but was prevented by the absence of platelet GPVI. Fibrinogen was deposited in glomerular capillaries via a partially intercellular adhesion molecule 1 (ICAM-1)-dependent mechanism, and inhibition of alpha(IIb)beta(3), fibrinogen and ICAM-1 inhibited platelet recruitment. Notably, neutrophil depletion also reduced platelet accumulation, indicating a cooperative interaction underlying recruitment of platelets and neutrophils. Finally, using bone marrow chimeras to restrict expression of P-selectin to platelets or endothelial cells, platelet but not endothelial P-selectin was required for glomerular leukocyte recruitment. Together these data indicate that platelet recruitment in this model is dependent on the combined actions of GPVI and the alpha(IIb)beta(3)/fibrinogen/ICAM-1 pathway and that platelet P-selectin is crucial for subsequent leukocyte recruitment.

A microfluidics device to monitor platelet aggregation dynamics in response to strain rate micro-gradients in flowing blood.

This paper reports the development of a platform technology for measuring platelet function and aggregation based on localized strain rate micro-gradients. Recent experimental findings within our laboratories have identified a key role for strain rate micro-gradients in focally triggering initial recruitment and subsequent aggregation of discoid platelets at sites of blood vessel injury. We present the design justification, hydrodynamic characterization and experimental validation of a microfluidic device incorporating contraction-expansion geometries that generate strain rate conditions mimicking the effects of pathological changes in blood vessel geometry. Blood perfusion through this device supports our published findings of both in vivo and in vitro platelet aggregation and confirms a critical requirement for the coupling of blood flow acceleration to downstream deceleration for the initiation and stabilization of platelet aggregation, in the absence of soluble platelet agonists. The microfluidics platform presented will facilitate the detailed analysis of the effects of hemodynamic parameters on the rate and extent of platelet aggregation and will be a useful tool to elucidate the hemodynamic and platelet mechano-transduction mechanisms, underlying this shear-dependent process.

A shear gradient-dependent platelet aggregation mechanism drives thrombus formation.

Platelet aggregation at sites of vascular injury is essential for hemostasis and arterial thrombosis. It has long been assumed that platelet aggregation and thrombus growth are initiated by soluble agonists generated at sites of vascular injury. By using high-resolution intravital imaging techniques and hydrodynamic analyses, we show that platelet aggregation is primarily driven by changes in blood flow parameters (rheology), with soluble agonists having a secondary role, stabilizing formed aggregates. We find that in response to vascular injury, thrombi initially develop through the progressive stabilization of discoid platelet aggregates. Analysis of blood flow dynamics revealed that discoid platelets preferentially adhere in low-shear zones at the downstream face of forming thrombi, with stabilization of aggregates dependent on the dynamic restructuring of membrane tethers. These findings provide insight into the prothrombotic effects of disturbed blood flow parameters and suggest a fundamental reinterpretation of the mechanisms driving platelet aggregation and thrombus growth.

Identification of a fibrin-independent platelet contractile mechanism regulating primary hemostasis and thrombus growth.

A fundamental property of platelets is their ability to transmit cytoskeletal contractile forces to extracellular matrices. While the importance of the platelet contractile mechanism in regulating fibrin clot retraction is well established, its role in regulating the primary hemostatic response, independent of blood coagulation, remains ill defined. Real-time analysis of platelet adhesion and aggregation on a collagen substrate revealed a prominent contractile phase during thrombus development, associated with a 30% to 40% reduction in thrombus volume. Thrombus contraction developed independent of thrombin and fibrin and resulted in the tight packing of aggregated platelets. Inhibition of the platelet contractile mechanism, with the myosin IIA inhibitor blebbistatin or through Rho kinase antagonism, markedly inhibited thrombus contraction, preventing the tight packing of aggregated platelets and undermining thrombus stability in vitro. Using a new intravital hemostatic model, we demonstrate that the platelet contractile mechanism is critical for maintaining the integrity of the primary hemostatic plug, independent of thrombin and fibrin generation. These studies demonstrate an important role for the platelet contractile mechanism in regulating primary hemostasis and thrombus growth. Furthermore, they provide new insight into the underlying bleeding diathesis associated with platelet contractility defects.