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.     

Amyloid β aggregation induces human brain microvascular endothelial cell death with abnormal actin organization

Cerebral amyloid angiopathy (CAA) is a disease in which amyloid β (Aβ) is deposited on the walls of blood vessels in the brain, making those walls brittle and causing cerebral hemorrhage. However, the mechanism underlying its onset is not well understood. The aggregation and accumulation of Aβ cause the occlusion and fragility of blood vessels due to endothelial cell damage, breakdown of the blood-brain barrier, and replacement with elements constituting the blood vessel wall. In this study, we observed the effect of Aβ on human primary brain microvascular endothelial cells (hBMECs) in real-time using quantum dot nanoprobes to elucidate the mechanism of vascular weakening by Aβ. It was observed that Aβ began to aggregate around hBMECs after the start of incubation and that the cells were covered with aggregates. Aβ aggregates firmly anchored the cells on the plate surface, and eventually suppressed cell motility and caused cell death. Furthermore, Aβ aggregation induced the organization of abnormal actin, resulting in a significant increase in intracellular actin dots over 10 μm². These results suggest that the mechanism by which Aβ forms a fragile vessel wall is as follows: Aβ aggregation around vascular endothelial cells anchors them to the substrate, induces abnormal actin organization, and leads to cell death.     

Finite platelet size could be responsible for platelet margination effect

Blood flows through vessels as a segregated suspension. Erythrocytes distribute closer to the vessel axis, whereas platelets accumulate near vessel walls. Directed platelet migration to the vessel walls promotes their hemostatic function. The mechanisms underlying this migration remain poorly understood, although various hypotheses have been proposed to explain this phenomenon (e.g., the available volume model and the drift-flux model). To study this issue, we constructed a mathematical model that predicts the platelet distribution profile across the flow in the presence of erythrocytes. This model considers platelet and erythrocyte dimensions and assumes an even platelet distribution between erythrocytes. The model predictions agree with available experimental data for near-wall layer margination using platelets and platelet-modeling particles and the lateral migration rate for these particles. Our analysis shows that the strong expulsion of the platelets from the core to the periphery of the blood vessel may mainly arise from the finite size of the platelets, which impedes their positioning in between the densely packed erythrocytes in the core. This result provides what we believe is a new insight into the rheological control of platelet hemostasis by erythrocytes.     

Kindlin-3 is essential for integrin activation and platelet aggregation

Integrin-mediated platelet adhesion and aggregation are essential for sealing injured blood vessels and preventing blood loss, and excessive platelet aggregation can initiate arterial thrombosis, causing heart attacks and stroke. To ensure that platelets aggregate only at injury sites, integrins on circulating platelets exist in a low-affinity state and shift to a high-affinity state (in a process known as integrin activation or priming) after contacting a wounded vessel. The shift is mediated through binding of the cytoskeletal protein Talin to the beta subunit cytoplasmic tail. Here we show that platelets lacking the adhesion plaque protein Kindlin-3 cannot activate integrins despite normal Talin expression. As a direct consequence, Kindlin-3 deficiency results in severe bleeding and resistance to arterial thrombosis. Mechanistically, Kindlin-3 can directly bind to regions of beta-integrin tails distinct from those of Talin and trigger integrin activation. We have therefore identified Kindlin-3 as a novel and essential element for platelet integrin activation in hemostasis and thrombosis.     

P2 receptors and platelet function

Following vessel wall injury, platelets adhere to the exposed subendothelium, become activated and release mediators such as TXA₂ and nucleotides stored at very high concentration in the so-called dense granules. Released nucleotides and other soluble agents act in a positive feedback mechanism to cause further platelet activation and amplify platelet responses induced by agents such as thrombin or collagen. Adenine nucleotides act on platelets through three distinct P2 receptors: two are G protein-coupled ADP receptors, namely the P2Y₁ and P2Y₁₂ receptor subtypes, while the P2X₁ receptor ligand-gated cation channel is activated by ATP. The P2Y₁ receptor initiates platelet aggregation but is not sufficient for a full platelet aggregation in response to ADP, while the P2Y₁₂ receptor is responsible for completion of the aggregation to ADP. The latter receptor, the molecular target of the antithrombotic drugs clopidogrel, prasugrel and ticagrelor, is responsible for most of the potentiating effects of ADP when platelets are stimulated by agents such as thrombin, collagen or immune complexes. The P2X₁ receptor is involved in platelet shape change and in activation by collagen under shear conditions. Each of these receptors is coupled to specific signal transduction pathways in response to ADP or ATP and is differentially involved in all the sequential events involved in platelet function and haemostasis. As such, they represent potential targets for antithrombotic drugs.     

Oxidative stress in haemostasis

The normal hemostatic mechanisms consist of a balance between hemorrhage and thrombosis that is achieved through the interaction of the blood vessels, blood platelets, the coagulation and fibrinolytic factors. The vascular endothelium sustains the balance between prevention and stimulation of platelet activation, thrombogenesis and fibrinolysis and between vasoconstriction and vasodilatation. Endothelial dysfunction associated with different cardiovascular diseases is related to the local formation of reactive oxygen/nitrogen species, mainly peroxynitrite that is produced in a rapid reaction between nitric oxide and superoxide anion. Reactive oxygen/nitrogen species induce changes in the structure and function in hemostatic elements. Proteins and lipids are major initial targets in endothelial cells, blood platelets and plasma. Reaction of reactive oxygen species and nitrogen species, including peroxynitrite, with cellular proteins can lead to nitration of aromatic amino acid residues, oxidation of thiol groups and conversion of some amino acid residues into carbonyl derivative. Oxidative/nitrative modifications of platelet proteins may induce changes of their signaling and haemostatic function (activation). Peroxynitrite also causes oxidation and nitration of fibrinogen--a key protein in coagulation cascade and plasminogen (the main protein of fibrinolysisprocess) changing their hemostatic functions. Oxidative/nitrative modifications of different components of haemostasis system have been observed in several cardiovascular diseases.     

Angiogenesis inhibitors found within the haemostasis pathway

Angiogenesis, the development of new blood vessels from the existing vasculature, and haemostasis, the coagulation cascade leading to formation of a clot, are among the most consistent host responses associated with cancer. Importantly, these two pathways interrelate, with blood coagulation and fibrinolysis influencing tumor angiogenesis directly, thereby contributing to tumor growth. Moreover, many endogenous inhibitors of angiogenesis are found within platelets or harboured as cryptic fragments of haemostatic proteins. In this review we outline ways in which angiogenesis is coordinated and regulated by haemostasis in human cancer. Then we detail the experimental and pre-clinical evidence for the ability of many of these endogenous proteins to inhibit tumor angiogenesis and thus their potential to be anti-cancer agents, with particular reference to any clinical trials.     

Inherited abnormalities of platelet glycoproteins

Glanzmann's thrombasthenia and the Bernard-Soulier syndrome are inherited blood disorders characterized by abnormalities in different aspects of platelet function during haemostasis. Platelets from patients with thrombasthenia do not aggregate in response to the normal physiological platelet aggregation inducing stimuli, while Bernard-Soulier platelet have a reduced capacity to adhere to exposed subendothelium. Deficiencies of different membrane glycoproteins have been located in the platelets of both disorders and suggest specific roles for membrane glycoproteins in different aspects of platelet function.     

Evidence for the dependence of arterial haemostasis on ADP

The possible involvement of adenosine diphosphate (ADP) in haemostatic platelet aggregation was investigated by determining the duration of primary haemorrhage as standardized bleeding times from punctures of small mesenteric arteries in anaesthetized rats. The bleeding times were highly significantly increased by infusing into the mesenteric arterial blood flowing towards the punctures either the nucleotide-dephosphorylating enzyme apyrase or the ADP-receptor antagonists ATP, adenosine 5'-(beta,gamma-methylene)triphosphonate (AMP-PCP) or 2-methylthioadenosine 5'-(beta,gamma-methylene)triphosphonate (2-MeS-AMP-PCP). The increases in bleeding times could not be accounted for by local vasodilator effects of the agents. It is concluded that the presence of ADP through local release and/or formation at sites of vascular injury contributes significantly to haemostasis, presumably by accelerating platelet aggregation.     

Platelet interactions in thrombosis

Patho/physiological platelet aggregate (thrombus) formation is initiated by engagement of platelet surface receptors, glycoprotein (GP)Ib-IX-V and GPVI that bind von Willebrand factor or collagen. Although beneficial in response to vascular injury by preventing blood loss (haemostasis), platelet aggregation in a sclerotic coronary artery or other diseased blood vessel (thrombosis) can cause thrombotic diseases like heart attack and stroke. At the molecular level, ligand interactions with GPIb-IX-V or GPVI trigger signalling responses, including elevation of cytosolic Ca2+, dissociation of calmodulin from their cytoplasmic domains, cytoskeletal actin-filament rearrangements, activation of src-family kinases or PI 3-kinase, and 'inside-out' activation of the integrin, alphaIIbbeta3 (GPIIb-llla), that binds von Willebrand factor or fibrinogen and mediates platelet aggregation. Furthermore, emerging evidence supports a topographical co-association of these receptors of the leucine-rich repeat family (GPIb-IX-V) and immunoglobulin superfamily (GPVI) in an adhesive cluster or 'adhesosome'. This arrangement may underlie common mechanisms of initiating thrombus formation in haemostasis or thrombotic disease.     

Identification of the disulphide bonds in human platelet glycocalicin

The glycoprotein Ib/IX complex on platelets is responsible for the first stage of haemostasis as an essential component in the primary adhesion of platelets to damaged vessel walls. Glycocalicin is the extracellular part of platelet glycoprotein Ib alpha and contains the von Willebrand factor and thrombin binding sites. Disulphide bonds are implicated in the von Willebrand binding site and studies with peptides point towards a region of glycocalicin with four cysteines as containing the binding sites for both von Willebrand factor and thrombin. The position and linkage of these two disulphide bonds are now determined to be 209-248 and 211-264 and the relevance of this double-loop structure for glycoprotein Ib/IX function is discussed.     

Interference with the Host Haemostatic System by Schistosomes

Schistosomes, parasitic flatworms that cause the tropical disease schistosomiasis, are still a threat. They are responsible for 200 million infections worldwide and an estimated 280,000 deaths annually in sub-Saharan Africa alone. The adult parasites reside as pairs in the mesenteric or perivesicular veins of their human host, where they can survive for up to 30 years. The parasite is a potential activator of blood coagulation according to Virchow''s triad, because it is expected to alter blood flow and endothelial function, leading to hypercoagulability. In contrast, hepatosplenic schistosomiasis patients are in a hypocoagulable and hyperfibrinolytic state, indicating that schistosomes interfere with the haemostatic system of their host. In this review, the interactions of schistosomes with primary haemostasis, secondary haemostasis, fibrinolysis, and the vascular tone will be discussed to provide insight into the reduction in coagulation observed in schistosomiasis patients.Interference with the haemostatic system by pathogens is a common mechanism and has been described for other parasitic worms, bacteria, and fungi as a mechanism to support survival and spread or enhance virulence. Insight into the mechanisms used by schistosomes to interfere with the haemostatic system will provide important insight into the maintenance of the parasitic life cycle within the host. This knowledge may reveal new potential anti-schistosome drug and vaccine targets. In addition, some of the survival mechanisms employed by schistosomes might be used by other pathogens, and therefore, these mechanisms that interfere with host haemostasis might be a broad target for drug development against blood-dwelling pathogens. Also, schistosome antithrombotic or thrombolytic molecules could form potential new drugs in the treatment of haemostatic disorders.     

Rheological factors in platelet - vessel wall interactions

Rheological aspects of platelet-vessel wall interactions involve cell-cell encounters, platelet - vessel wall encounters and platelet-thrombus interactions. The cell-cell encounters are usually caused by convection of cells in shear flows rather than by Brownian motion; this is important in aggregation and in the enhancement of the diffusion of platelets by red cell motion. Platelet - vessel wall interactions can involve transient adhesion (lasting from a fraction of a second to a few minutes) as well as more permanent adhesion. Reaction rates between platelets and walls are generally very small except on damaged vessels and some artificial surfaces. Ultra-filtration through the vessel wall affects cell-wall interactions. Rheological analyses of thrombus formation have been made and shown interesting relations to experimental observations. Some experimental results have indicated that platelets are capable of reacting within a small fraction of a second. Red cells may act as mechanoreceptors for increases in shear rate and facilitate the speed of response of platelets. Surface geometrical forms such as bumps and cavities tend to prolong residence times and facilitate thrombus formation.     

Inhibition of microsurgical thrombosis by the platelet glycoprotein IIb/IIIa antagonist SR121566A

Despite major improvements in tools and significant refinements of techniques, microsurgical anastomosis still carries a significant risk of failure due to microvascular thrombosis. The key to improving the success of microvascular surgery may lie in the pharmacologic control of thrombus formation. Central to pathologic arterial thrombosis are platelets. Glycoprotein IIb/IIIa is a highly abundant platelet surface receptor that plays a major role in platelet aggregation by binding platelets to each other through the coagulation factor fibrinogen. To explore the ability of antithrombotic agents to prevent microvascular thrombosis, a rabbit ear artery model was used in which a standardized arterial injury results in predictable thrombus formation. This model was used to examine whether SR121566A, a specific and potent glycoprotein IIb/IIIa inhibitor, can successfully prevent microsurgical thrombosis.Using a coded, double-blind experimental design, 20 rabbits (40 arteries) were assigned to four treatment groups: (1) saline injection (n = 10), (2) acetylsalicylic acid 10 mg/kg (n = 10), (3) heparin 0.5 mg/kg bolus with subsequent intermittent boluses of 0.25 mg/kg every 30 minutes (n = 10), and (4) SR121566A 2 mg/kg bolus (n = 10). After vessel damage and clamp release, arteries were assessed for patency at 5, 30, and 120 minutes by the Acland refill test. Coagulation assays, in vivo bleeding times, and ex vivo platelet aggregation studies were also conducted. Scanning electron microscopy was used to examine mural thrombus composition.A significant, fourfold increase in vessel patency following administration of SR121566A over saline control (80 percent versus 20 percent patency, respectively, at 35 minutes after reperfusion, p < 0.01) was noted. This was correlated with marked inhibition of ex vivo platelet aggregation. This antiplatelet treatment did not prolong coagulation assays (mean international normalized ratio: saline, 0.66 +/- 0.04; SR121566A, 0.64 +/- 0.03; mean thromboplastin time: saline, 19.63 +/- 0.67; SR121566A, 17.87 +/- 3.27) and bleeding times (mean bleeding time: saline, 42 +/- 4; SR121566A, 48 +/- 6). Scanning electron microscopy demonstrated extensive platelet and fibrin deposition in control vessel thrombi. In contrast, thrombi from SR121566A-treated vessels demonstrated predominance of fibrin with few platelets when examined under scanning electron microscopy.Administration of SR121566A was associated with a significant increase in vessel patency, without deleterious effects on coagulation assays or bleeding times. The increase in vessel patency was correlated with inhibition of platelet aggregation and decreased platelet deposition, as demonstrated by scanning electron microscopy. Glycoprotein IIb/IIIa antagonists represent a new class of anti-platelet agents that may be suited for inhibiting microsurgical thrombosis. This study supports further investigation into the use of these agents in microsurgery.     

Spatial and temporal role of the apelin/APJ system in the caliber size regulation of blood vessels during angiogenesis

Blood vessels change their caliber to adapt to the demands of tissues or organs for oxygen and nutrients. This event is mainly organized at the capillary level and requires a size-sensing mechanism. However, the molecular regulatory mechanism involved in caliber size modification in blood vessels is not clear. Here we show that apelin, a protein secreted from endothelial cells under the activation of Tie2 receptor tyrosine kinase on endothelial cells, plays a role in the regulation of caliber size of blood vessel through its cognate receptor APJ, which is expressed on endothelial cells. During early embryogenesis, APJ is expressed on endothelial cells of the new blood vessels sprouted from the dorsal aorta, but not on pre-existing endothelial cells of the dorsal aorta. Apelin-deficient mice showed narrow blood vessels in intersomitic vessels during embryogenesis. Apelin enhanced endothelial cell proliferation in the presence of vascular endothelial growth factor and promoted cell-to-cell aggregation. These results indicated that the apelin/APJ system is involved in the regulation of blood vessel diameter during angiogenesis.     

Platelet activation receptor CLEC-2 regulates blood/lymphatic vessel separation by inhibiting proliferation, migration, and tube formation of lymphatic endothelial cells

The platelet activation receptor CLEC-2 plays crucial roles in thrombosis/hemostasis, tumor metastasis, and lymphangiogenesis, although its role in thrombosis/hemostasis remains controversial. An endogenous ligand for CLEC-2, podoplanin, is expressed in lymphatic endothelial cells (LECs). We and others have reported that CLEC-2-deficiency is lethal at mouse embryonic/neonatal stages associated with blood-filled lymphatics, indicating that CLEC-2 is essential for blood/lymphatic vessel separation. However, its mechanism, and whether CLEC-2 in platelets is necessary for this separation, remains unknown. We found that specific deletion of CLEC-2 from platelets leads to the misconnection of blood/lymphatic vessels. CLEC-2(+/+) platelets, but not by CLEC-2(-/-) platelets, inhibited LEC migration, proliferation, and tube formation but had no effect on human umbilical vein endothelial cells. Additionally, supernatants from activated platelets significantly inhibited these three functions in LECs, suggesting that released granule contents regulate blood/lymphatic vessel separation. Bone morphologic protein-9 (BMP-9), which we found to be present in platelets and released upon activation, appears to play a key role in regulating LEC functions. Only BMP-9 inhibited tube formation, although other releasates including transforming growth factor-β and platelet factor 4 inhibited proliferation and/or migration. We propose that platelets regulate blood/lymphatic vessel separation by inhibiting the proliferation, migration, and tube formation of LECs, mainly because of the release of BMP-9 upon activation by CLEC-2/podoplanin interaction.     

Small aggregates of platelets can be detected sensitively by a flow cytometer equipped with an imaging device: mechanisms of epinephrine-induced aggregation and antiplatelet effects of beraprost

BACKGROUND: Although cross-talks between platelets and other blood cells are important in vivo, laboratory platelet aggregation tests have been performed mainly with the use of platelet-rich plasma (PRP) as samples. Methods that enable an efficient and sensitive detection of platelet aggregates in whole blood are being developed. METHODS: A flow cytometer equipped with an imaging device, the flow imaging cytometer 2 (FIC2), was used to detect platelet aggregates in whole blood. RESULTS: The FIC2 provides a resolution that is high enough to differentiate platelet aggregates from single platelets or other blood cells. Epinephrine elicited platelet aggregate formation in hirudin plus argatroban-treated whole blood, but not in PRP. The reconstitution study revealed that a small amount of adenosine diphosphate (ADP) from erythrocytes may play an important role in epinephrine-induced platelet aggregation (in whole blood), through mediation of P2Y1 receptors. When the inhibitory effect of beraprost, an antiplatelet agent, on platelet aggregation was assessed, analysis of whole blood samples with FIC2 proved to be the most sensitive among the methods available. CONCLUSIONS: FIC2 is a promising device for detection of platelet aggregates in whole blood, with wide basic and clinical applications.     

Innervation of the microcirculatory bed of the human trigeminal nerve]

The investigation of film preparations and histological sections of human trigeminal nerves impregnated with silver nitrate and treated after Gomori, Falck--Hillarp demonstrated a rich innervation in the intraneural blood vessels. The most various and complex interconnections of the neural structures were noted in arterioles and venules of the node capsule, epineurium and external layers of perineurium of the trigeminal nerve branches. On the vessel walls of these layers, neural plexus were revealed. Sensitive innervation of the neural blood vessels mainly performed by posvalent tissue-vascular receptors. In the walls of intraneural vessels, adrenergic and cholinergic neural plexus are revealed.     

Effect of ergoline derivatives on platelet and blood vessels]

The influence of dihydrogenated ergot alkaloids on vessels and blood platelets was studied in vitro and in vivo. At low concentrations they cause contraction of isolated vein strips via a stimulation of alpha-adrenoceptors, at higher concentrations they antagonize the action of noradrenaline on arteries and veins and inhibit the adrenaline-induced platelet aggregation in vitro. In volunteers, intravenous and subcutaneous administration of dihydroergotamine (Dihytamin) caused a decrease in blood volume of the capacitance vessels and inhibition of the adrenaline-potentiated platelet aggregation "ex vivo". The selective venoconstrictor and antiaggregating effects of dihydroergotamine are utilized in the postoperative prophylaxis of thrombosis.     

Increased platelet aggregability is associated with increased prostacyclin production by vessel walls in hypercholesterolemic rabbits

The influences of experimental hypercholesterolemia in the rabbit on platelet-vessel wall interactions have been studied by evaluating the aggregatory response of platelet rich plasma (PRP) to arachidonic acid (AA) stimulation and levels of 6-keto-PGF1 alpha in PRP from normal (N) and hypercholesterolemic (HC) animals prior and after perfusion through the corresponding aortas. In addition, the responses of N PRP to aggregation after perfusion through HC aortas and those of HC PRP perfused through N aortas, and the platelet response to the inhibitory effect of exogenous prostacyclin have been evaluated. The data indicate that in HC rabbits, on one side platelets are hyperreactive to AA and less sensitive to the inhibitory activity of prostacyclin and, on the other, the antiaggregatory activity and prostacyclin production of vessel walls is higher, suggesting compensatory mechanisms in the haemostatic balance.