Process Of Platelet Adhesion Research Articles

A Multiscale Model for Shear-Mediated Platelet Adhesion Dynamics: Correlating In Silico with In Vitro Results.

Platelet adhesion to blood vessel walls is a key initial event in thrombus formation in both vascular disease processes and prosthetic cardiovascular devices. We extended a deformable multiscale model (MSM) of flowing platelets, incorporating Dissipative Particle Dynamics (DPD) and Coarse-Grained Molecular Dynamics (CGMD) describing molecular-scale intraplatelet constituents and their interaction with surrounding flow, to predict platelet adhesion dynamics under physiological flow shear stresses. Binding of platelet glycoprotein receptor Ibα (GPIbα) to von Willebrand factor (vWF) on the blood vessel wall was modeled by a molecular-level hybrid force field and validated with in vitro microchannel experiments of flowing platelets at 30 dyne/cm2. High frame rate videos of flipping platelets were analyzed with a Semi-Unsupervised Learning System (SULS) machine learning-guided imaging approach to segment platelet geometries and quantify adhesion dynamics parameters. In silico flipping dynamics followed in vitro measurements at 15 and 45dyne/cm2 with high fidelity, predicting GPIbα-vWF bonding and debonding processes, distribution of bonds strength, and providing a biomechanical insight into initiation of the complex platelet adhesion process. The adhesion model and simulation framework can be further integrated with our established MSMs of platelet activation and aggregation to simulate initial mural thrombus formation on blood vessel walls.

In Vitro Measurement and Modeling of Platelet Adhesion on VWF-Coated Surfaces in Channel Flow

The process of platelet adhesion is initiated by glycoprotein (GP)Ib and GPIIbIIIa receptors on the platelet surface binding with von Willebrand factor on the vascular walls. This initial adhesion and detachment of a single platelet is a complex process that involves multiple bonds forming and breaking and is strongly influenced by the surrounding blood-flow environment. In addition to bond-level kinetics, external factors such as shear rate, hematocrit, and GPIb and GPIIbIIIa receptor densities have also been identified as influencing the platelet-level rate constants in separate studies, but this still leaves a gap in understanding between these two length scales. In this study, we investigate the fundamental relationship of the dynamics of platelet adhesion, including these interrelating factors, using a coherent strategy. We build a, to our knowledge, novel and computationally efficient multiscale model accounting for multibond kinetics and hydrodynamic effects due to the flow of a cellular suspension. The model predictions of platelet-level kinetics are verified by our microfluidic experiments, which systematically investigate the role of each external factor on platelet adhesion in an in vitro setting. We derive quantitative formulas describing how the rates of platelet adhesion, translocation, and detachment are defined by the molecular-level kinetic constants, the local platelet concentration near the reactive surface determined by red-blood-cell migration, the platelet effective reactive area due to its tumbling motion, and the platelet surface receptor density. Furthermore, if any of these aspects involved have abnormalities, e.g., in a disease condition, our findings also have clinical relevance in predicting the resulting change in the adhesion dynamics, which is essential to hemostasis and thrombosis.

Haemostatic role of intermediate filaments in adhered platelets: Importance of the membranous system stability

The role of platelets in coagulation and the haemostatic process was initially suggested two centuries ago, and under appropriate physiological stimuli, these undergo abrupt morphological changes, attaching and spreading on damaged endothelium, preventing bleeding. During the adhesion process, platelet cytoskeleton reorganizes generating compartments in which actin filaments, microtubules, and associated proteins are arranged in characteristic patterns mediating crucial events, such as centralization of their organelles, secretion of granule contents, aggregation with one another to form a haemostatic plug, and retraction of these aggregates. However, the role of Intermediate filaments during the platelet adhesion process has not been explored. J. Cell. Biochem. 114: 2050-2060, 2013. © 2013 Wiley Periodicals, Inc.

Multiscale Modeling of Platelet Adhesion and Thrombus Growth

Our hemostatic system, when called to action, depends on the complex arrangement of a tightly regulated and extensive network of molecules with versatile functionality. Experimental methods have demonstrated marked improvement through enhanced condition-control and monitoring. However, this approach continues to provide limited explanations of the role of individual elements or of a specific component within the entire system. To fill this void, multiscale simulations based on high throughput computing and comprehensive mathematical models are showing their strength in not only revealing hidden physiological mechanisms but also predicting pharmacological/phenotypical outcome in hemostasis reactions based on quantitative analysis. In this review article, we present up-to-date computational methods that simulate the process of platelet adhesion and thrombus growth, compare and summarize their advantages and drawbacks, verify their predictive power, and project their future directions. We provide an in-depth summary of one such computational method-Platelet Adhesive Dynamics (PAD)-and discuss its application in simulating platelet aggregation and thrombus development.

Modelling of thrombus growth and growth stop in flow by the method of dissipative particle dynamics

Platelet aggregation at the site of the vascular injury leads to the formation of a hemostatic plug covering the injury site, or a thrombus in the pathological case. The mechanisms that control clot growth or lead to growth arrest are not yet completely understood. In order to study these mechanisms theoretically, we use the Dissipative Particle Dynamics method, which allows us to model individual platelets in the flow and in the clot. The model takes into account different stages of the platelet adhesion process. First, a platelet is captured reversibly by the aggregate, then it is activated and adheres firmly, becoming a part of its core.We suggest that the core of the clot is composed of platelets unable to attach new platelets from the flow due to their activation by thrombin and/or wrapping by the fibrin mesh. The simulations are in a good agreement with the experimental results [9]. Modelling shows that stopping the growth of a hemostatic plug (and thrombus) may result from its exterior part being removed by the flow and exposed its non-adhesive core to the flow.

Actin filaments and microtubule dual‐granule transport in human adhered platelets: the role of α‐dystrobrevins

Upon activation with physiological stimuli, human platelets undergo morphological changes, centralizing their organelles and secreting effector molecules at the site of vascular injury. Previous studies have indicated that the actin filaments and microtubules of suspension-activated platelets play a critical role in granule movement and exocytosis; however, the participation of these cytoskeleton elements in adhered platelets remains unexplored. alpha- and beta-dystrobrevin members of the dystrophin-associated protein complex in muscle and non-muscle cells have been described as motor protein receptors that might participate in the transport of cellular components in neurons. Recently, we characterized the expression of dystrobrevins in platelets; however, their functional diversity within this cellular model had not been elucidated. The present study examined the contribution of actin filaments and microtubules in granule trafficking during the platelet adhesion process using cytoskeleton-disrupting drugs, quantification of soluble P-selectin, fluorescence resonance transfer energy analysis and immunoprecipitation assays. Likewise, we assessed the interaction of alpha-dystrobrevins with the ubiquitous kinesin heavy chain. Our results strongly suggest that microtubules and actin filaments participate in the transport of alpha and dense granules in the platelet adhesion process, during which alpha-dystrobrevins play the role of regulatory and adaptor proteins that govern trafficking events.

Inhibitors of Platelet Adhesion

Platelet aggregation, in fundamental terms, is considered a biological end point that contributes to the occurrence of clinical events among patients with advanced atherosclerotic coronary artery disease. Acute coronary syndrome, including non–ST elevation myocardial infarction (NSTEMI) and ST elevation myocardial infarction (STEMI), accounts for upward of 733 000 hospital admissions yearly in the United States.1 The primary pathophysiological mechanism responsible for the majority of acute coronary syndromes is endothelial plaque disruption with and subsequent platelet adhesion, activation, and thrombus formation.2 The end result is formation of thrombus within a coronary artery, leading to subtotal vessel occlusion with NSTEMI and complete occlusion of the artery with STEMI. In this review, we provide a contemporary view of platelet adhesion as a highly coordinated and teleologically conserved process achieved by surface receptors, protein ligands, and matrix proteins operating at the platelet–subendothelium interface. We also discuss drugs in development, including monoclonal antibodies, inhibitory peptides, and oligonucleotides; preclinical data; and, where available, clinical trial results, highlighting the potential translation of fundamental constructs in platelet biology to patient care. Platelets are derived from a hematopoietic bone marrow stem cell precursor.3 Through a highly conserved program of cellular differentiation, this stem cell precursor becomes a megakaryocyte at a rate of approximately 100 000 000 megakaryocytes produced per day.3 Subsequently, each individual megakaryocyte will give rise to approximately 500 platelets via additional developmental steps within the bone marrow that involve a proplatelet intermediate stage.3 Once released into circulation, a mature platelet has an expected life span of 7 to 10 days.3 Platelet maturation and development involve the expression of receptors on the platelet cell surface. These receptors facilitate platelet adhesion and activation, and they promote thrombus development through receptor–ligand interactions, with several ligands expressed on the surface of endothelial cells, within the …

血小板粘着反応におけるvon Willebrand因子のドメイン構造とそのin vitroモジュレーター

血小板粘着反応におけるvon Willebrand因子のドメイン構造とそのin vitroモジュレーター

β‐Dystroglycan modulates the interplay between actin and microtubules in human‐adhered platelets

To maintain the continuity of an injured blood vessel, platelets change shape, secrete granule contents, adhere, aggregate, and retract in a haemostatic plug. Ordered arrays of microtubules, microfilaments, and associated proteins are responsible for these platelet responses. In full-spread platelets, microfilament bundles in association with other cytoskeleton proteins are anchored in focal contacts. Recent studies in migrating cells suggest that co-ordination and direct physical interaction of microtubules and actin network modulate adhesion development. In platelets, we have proposed a feasible association between these two cytoskeletal systems, as well as the participation of the dystrophin-associated protein complex, as part of the focal adhesion complex. The present study analysed the participation of microtubules and actin during the platelet adhesion process. Confocal microscopy, fluorescence resonance transfer energy and immunoprecipitation assays were used to provide evidence of a cross-talk between these two cytoskeletal systems. Interestingly, beta-dystroglycan was found to act as an interplay protein between actin and microtubules and an additional communication between these two cytoskeleton networks was maintained through proteins of focal adhesion complex. Altogether our data are indicative of a dynamic co-participation of actin filaments and microtubules in modulating focal contacts to achieve platelet function.

Role of dystrophins and utrophins in platelet adhesion process

Platelets are crucial at the site of vascular injury, adhering to the sub-endothelial matrix through receptors on their surface, leading to cell activation and aggregation to form a haemostatic plug. Platelets display focal adhesions as well as stress fibres to contract and facilitate expulsion of growth and pro-coagulant factors contained in the granules and to constrict the clot. The interaction of F-actin with different actin-binding proteins determines the properties and composition of the focal adhesions. Recently, we demonstrated the presence of dystrophin-associated protein complex corresponding to short dystrophin isoforms (Dp71d and Dp71) and the uthophin gene family (Up400 and Up71), which promote shape change, adhesion, aggregation, and granule centralisation. To elucidate participation of both complexes during the platelet adhesion process, their potential association with integrin beta-1 fraction and the focal adhesion system (alpha-actinin, vinculin and talin) was evaluated by immunofluorescence and immunoprecipitation assays. It was shown that the short dystrophin-associated protein complex participated in stress fibre assembly and in centralisation of cytoplasmic granules, while the utrophin-associated protein complex assembled and regulated focal adhesions. The simultaneous presence of dystrophin and utrophin complexes indicates complementary structural and signalling mechanisms to the actin network, improving the platelet haemostatic role.

The study of anaemia-related haemostasis impairment in haemodialysis patients by in vitro closure time test

It is known that anaemia in haemodialysis patients could contribute to haemostasis impairment. However, the precise relation between the degree of anaemia and the degree of haemostasis impairment is not known, nor the optimal level of hematocrit above which anaemia no longer disturbs haemostasis. Our study addresses these clinically relevant questions by employing in vitro closure time test, a new method in which the process of platelet adhesion and aggregation following vascular injury is simulated in vitro in samples of whole blood. We studied 63 haemodialysis patients, with 30 age-matched, healthy controls. Results show that patients with hematocrit below 0.32 (N=28) had significantly impaired primary haemostasis, in contrast to patients with hematocrit above 0.32 (N=35), as measured by both types of closure time test. A significant negative association was found between hematocrit values and closure time (CEPI cartridges: rho=-0.41, p<0.001; CADP cartridges: rho=-0.47, p<0.001). A multiple logistic regression model for predicting prolonged closure time confirmed this finding. Nonparametric curve fitting enabled estimation of the level of hematocrit at which the values of in vitro closure time in haemodialysis patients do not differ from those in the controls at approximately 0.35. ROC analysis confirmed this to be the optimal threshold for predicting prolonged closure time for both cartridges. By using in vitro closure time test, we confirmed that anaemia correlates with the severity of haemostasis impairment. We estimated the target level of hematocrit above which anaemia no longer affects haemostasis to be about 0.35. These new results (and new assay) appear to have clinical value for treating haemodialysis patients.

Platelet retention test Homburg (RTH) and drug monitoring of platelet adhesive properties of von Willebrand factor

Platelet plug formation is initiated by the process of platelet adhesion, mainly mediated by the von Wille-brand factor (VWF). Therefore, apart from established criteria the platelet adhesion property is a further criterion to determine VWF e. g. in diagnosis and treatment of von Willebrand disease (VWD). The new platelet retention test Homburg (RTH) is designed to close this gap. It is characterized by its non-thrombogenic filter with interconnecting pores, which retains platelets from blood when pressed through this filter due to the resulting shear stress. The RTH, in particular, proved to be highly sensitive in detecting the platelet adhesive property of VWF after its release from endogenous storage sites by desmopressin or infusion in VWD patients or its supplementation in vitro.

Plättchen-Retentionstest Homburg

ZusammenfassungDie Plättchenpfropfbildung wird durch den Prozess der Plättchenadhäsion eingeleitet, der hauptsächlich vom von-Willebrand-Faktor (VWF) vermittelt wird. Neben etablierten Parametern ist daher die Fähigkeit zur Plättchenadhäsion ein weiteres Kriterium zur VWF-Bestimmung, z. B. in der Diagnostik und Therapie der von-Willebrand-Krankheit. Der neue Plättchen-Retentionstest Homburg (RTH) soll helfen, diese Lücke zu schließen. Er ist charakterisiert durch seinen nicht thrombogenen Filter mit interkonnektierenden Poren, der die Plättchen aus dem Blut aufgrund der Scherkraft zurückhält, die beim Strömen durch den Filter entsteht. Der RTH erwies sich als sehr empfindlich zur Erfassung der plättchenadhäsiven Eigenschaft des VWF nach dessen Freisetzung aus endogenen Speichern durch Desmopressin oder VWF-Infusion bei Patienten mit von-Willebrand-Krankheit sowie bei entsprechenden In-vitro-Versuchen.

Utility of in vitro closure time test for evaluating platelet-related primary hemostasis in dialysis patients

Background:The platelet aggregation and skin bleeding time (SBT) tests currently used for assessment of hemostasis impairment in dialysis patients have important disadvantages. The authors explored the utility of a novel in vitro closure time test (PFA-100, platelet function analyzer) in which the process of platelet adhesion and aggregation after vascular injury is simulated in vitro in dialysis patients. Methods:Thirty-four long-term dialysis patients were included in the study with 30 healthy volunteers as the control group. In vitro closure time was compared with results from the platelet aggregation and SBT tests. Results:In vitro closure time identified more patients and fewer controls with hemostasis impairment. In the patient group, 60%, 40%, and 20%, and in the control group, 0%, 10% and 3% of persons were found to have hemostasis impairment as determined by in vitro closure time, platelet aggregation, and SBT, respectively. In addition, values for patients and controls were significantly different for in vitro closure time (P < 0.05) but not for platelet aggregation or SBT. Thus, closure time appears to be more sensitive and specific than the other 2 tests. No correlation was found between the 3 tests, either in patients or in controls. However, a high correlation (r = 0.73; P < 0.0001) was found between the 2 types of in vitro closure time test (collagen/epinephrine [CEPI] and collagen/adenosine diphosphate [CADP]) in patients and controls. Conclusion:These results indicate that in vitro closure time can be a useful test for detecting platelet-related primary hemostasis defects in dialysis patients.

Vascular biology of thrombosis: the role of platelet-vessel wall adhesion.

Arterial thrombosis is initiated after atherosclerotic plaque rupture. The ruptured plaque triggers a cascade of platelet-mediated events that result in the formation of a platelet-rich thrombus. Thrombus development ultimately leads to vessel occlusion, the precipitating event in most myocardial infarctions and many strokes. Arterial thrombosis involves a process of platelet adhesion, activation, and aggregation. Platelets first form a monolayer by adhering to blood vessel subendothelial matrix proteins (collagen and von Willebrand factor) via interactions with platelet membrane glycoprotein (GP) receptors. This interaction produces an intracellular signal that activates platelets to increase expression of a specific GP receptor, GPIIb/IIIa, with a high affinity for fibrinogen and other adhesion molecules. Binding of fibrinogen to adjacent platelets results in irreversible platelet aggregation and the formation of the platelet aggregate. Understanding of this process has led to considerable research into potential antiplatelet compounds. A number of effective antiplatelet drugs are now available, which affect different stages along the platelet adhesion/activation/aggregation pathway. These include aspirin, dipyridamole, and the adenosine diphosphate antagonists. Much effort is now focused on development of thrombin receptor antagonists. However, this is proving to be difficult. Considerable interest has surrounded a new class of platelet drugs, the GPIIb/IIIa receptor antagonists. These drugs have proved effective in short-term infusion trials. However, long-term studies of oral GPIIb/IIIa receptor antagonists have shown increases in mortality. It is believed that these antagonists may act as partial agonists and may increase the risk for thrombosis.

Shear-Dependent Rolling on von Willebrand Factor of Mammalian Cells Expressing the Platelet Glycoprotein Ib-IX-V Complex

Mural thrombi form on exposed arterial subendothelium by a two-step process of platelet adhesion and aggregation. At high shear stresses such as are found in stenotic arteries, both steps are mediated by von Willebrand factor (vWF). Platelets initially adhere on vWF affixed to the subendothelial matrix through the glycoprotein (GP) Ib-IX-V complex. To examine the role of the GP Ib-IX-V complex under dynamic conditions, we modeled initial platelet adhesion at shear stresses ranging from 2 to 40 dyn/cm2 using vWF-coated glass slides, mammalian cells expressing full or partial GP Ib-IX-V complexes, and a parallel plate flow chamber with phase contrast video microscopy and digital image processing. Mammalian cells expressing the full complex tethered and rolled on the vWF substrate, whereas control cells did not. The rolling was completely inhibited by the monoclonal GP Ib antibody, AK2, or the vWF antibody, 5D2, both shown previously to block vWF-dependent platelet aggregation. Other GP Ib antibodies, WM23 and SZ2, did not significantly change the number or mean velocity of rolling cells. At low levels of GP Ib surface expression, cells expressing the full complex rolled slower than cells expressing the complex without GP V, indicating that GP V strengthens the interactions with the vWF surface under these conditions. Preshearing vWF for 5 minutes at 40 dyn/cm2 immediately before introducing cells into the chamber did not significantly change the number or the mean velocity of rolling cells. Inhibiting sulfation of the tyrosine residues within the GP Ib subunit reduced the number but did not change the mean velocity of the rolling cells. Our results indicate that, under the conditions of these experiments, bonds between vWF and GP Ib constantly form and break under fluid shear stress. Additionally, our results suggest that GP Ib-IX-V complexes behave like selectin receptors in their ability to mediate smooth rolling while cells maintain continuous surface contact. Such a mechanism, in vivo, would allow platelets to slow down and eventually arrest on the blood vessel wall. The system described provides a valuable approach for investigating the structure-function relationship of individual receptors and ligands in the process of platelet adhesion and thrombosis.

Shear-Dependent Rolling on von Willebrand Factor of Mammalian Cells Expressing the Platelet Glycoprotein Ib-IX-V Complex

AbstractMural thrombi form on exposed arterial subendothelium by a two-step process of platelet adhesion and aggregation. At high shear stresses such as are found in stenotic arteries, both steps are mediated by von Willebrand factor (vWF). Platelets initially adhere on vWF affixed to the subendothelial matrix through the glycoprotein (GP) Ib-IX-V complex. To examine the role of the GP Ib-IX-V complex under dynamic conditions, we modeled initial platelet adhesion at shear stresses ranging from 2 to 40 dyn/cm2 using vWF-coated glass slides, mammalian cells expressing full or partial GP Ib-IX-V complexes, and a parallel plate flow chamber with phase contrast video microscopy and digital image processing. Mammalian cells expressing the full complex tethered and rolled on the vWF substrate, whereas control cells did not. The rolling was completely inhibited by the monoclonal GP Ibα antibody, AK2, or the vWF antibody, 5D2, both shown previously to block vWF-dependent platelet aggregation. Other GP Ibα antibodies, WM23 and SZ2, did not significantly change the number or mean velocity of rolling cells. At low levels of GP Ibα surface expression, cells expressing the full complex rolled slower than cells expressing the complex without GP V, indicating that GP V strengthens the interactions with the vWF surface under these conditions. Preshearing vWF for 5 minutes at 40 dyn/cm2 immediately before introducing cells into the chamber did not significantly change the number or the mean velocity of rolling cells. Inhibiting sulfation of the tyrosine residues within the GP Ibα subunit reduced the number but did not change the mean velocity of the rolling cells. Our results indicate that, under the conditions of these experiments, bonds between vWF and GP Ibα constantly form and break under fluid shear stress. Additionally, our results suggest that GP Ib-IX-V complexes behave like selectin receptors in their ability to mediate smooth rolling while cells maintain continuous surface contact. Such a mechanism, in vivo, would allow platelets to slow down and eventually arrest on the blood vessel wall. The system described provides a valuable approach for investigating the structure-function relationship of individual receptors and ligands in the process of platelet adhesion and thrombosis.

Interaction of Integrin αIIbβ3 with Multiple Fibrinogen Domains during Platelet Adhesion

We have investigated how modulation of integrin alpha IIb beta 3 function influences the mechanisms that initiate platelet thrombus formation onto surface-bound fibrinogen and isolated fibrinogen domains. Under stationary conditions and with full activation of platelets blocked by prostaglandin E1, the carboxyl-terminal gamma 400-411 sequence is necessary for establishing initial contact with the immobilized substrate. Molecules containing a single copy of this sequence, like the plasmin-generated fibrinogen fragment D, support platelet spreading, but the resulting attachment to the surface is loose and disrupted by minimal peeling force. In contrast, platelets adhere firmly to intact fibrinogen under the same conditions, suggesting that recognition of contact sites outside a single D domain can secure the firm interaction not supported by a single gamma 400-411 sequence. If platelets are activated, the gamma 400-411 sequence is no longer necessary to initiate the adhesion process but becomes sufficient, even as a single copy, to mediate stable surface attachment in the absence of shear stress. Under conditions of flow, however, intact fibrinogen but not fragment D can support adhesion, regardless of whether platelets have the potential to become activated or not. These results indicate the functional relevance of multiple fibrinogen domains during the initial stages of the platelet adhesion process.

Platelet adhesion at low shear rate: Study of a normal population

The use of oral contraceptives (OCs) has been associated with vascular complications. The mechanism(s) by which OCs predispose to thrombotic events remains unclear. Recent studies have demonstrated that postmenopausal (PM) women who take estrogen replacement therapy (ERT) have a decreased incidence of myocardial infarction compared to those who do not take ERT. This study was undertaken to determine if healthy individuals have differences in platelet adhesion depending on hormonal status. Men, PM women taking ERT, PM women not taking ERT, OC users, and premenopausal women not taking any medications were studied. Platelet studies were performed in a Hele-Shaw flow chamber at a low shear rate using platelet-rich plasma. The platelet adhesion process to subendothelial components: fibronectin, collagen I and collagen III was recorded using a 35 mm camera mounted on an inverted microscope. Photographs were taken at 30 second intervals for a total of 12 minutes and analyzed using a modified computer program which provided a numerical account of platelet adhesion. OC users had significantly higher platelet adherence to fibronectin, collagen I and collagen III compared to all other groups. All other study groups had similar platelet adhesion independent of hormonal status. These findings suggest that OCs cause increased platelet adhesion in some individuals and this may be one of the mechanisms by which OCs contribute to thrombotic events.

Morphological Study of Platelet Adhesion Dynamics Under Whole Blood Flow Conditions

A flow system consisting of a parallel-plate flow chamber mounted on the epifluorescence video microscope has been constructed to allow direct visualization of the entire platelet adhesion process under whole blood flow conditions. Adhered platelets with recorded adhesion history were individually identified and observed in detail using a scanning electron microscope. In this study we used cover glasses coated with fibrinogen, fibrin, or collagen as the testing surface. From experiments carried out at the surface shear rate of 445 s(-1), we found that (1) platelet adhesion was a dynamic process that involved attaching, detaching, relocation and transient contact; (2) platelets adhered to all three types of protein-coated surfaces with platelet adhesion on collagen being most unstable; (3) most of these adhered platelets immediately formed short pseudopods after surface contact; (4) platelets adhered to fibrinogen or fibrin were basically non-overlapping and they underwent further shape change with increasing number /length of pseudopods and increasing extent of cytoplasmic spreading; (5) on collagen-coated surface most incoming platelets attached to previously adhered platelets rather than to the collagen threads for blood-surface contact times longer than 30 s; (6) these platelets formed multicellular thrombi with largest thrombi located at about 0.2-0.4 mm from the upstream edge and (7) platelets in the thrombi formed numerous short pseudopods and started fusing with one another within 2 min. These observations show that platelet adhesion under blood flow is a complex and dynamic process and that adhered platelets undergo heterogeneous post-contact morphological changes. Moreover, our results indicate that fibrinogen and fibrin coatings are adhesive while collagen coating is most stimulatory to platelets.